[Histonet] RE: Histonet Digest, Vol 17, Issue 37

From:Malam Jacqueline

Hi
I had the same problem, especially with fatty tissues and found that
poly-l-lysine (SIGMA, code P-1524) used at 0.1% aqueous and spread onto a
slide as you would a blood smear worked much better.
Cheers
Jacqui
Lancaster

-----Original Message-----
From: histonet-request@lists.utsouthwestern.edu
[mailto:histonet-request@lists.utsouthwestern.edu] 
Sent: 26 April 2005 17:42
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 17, Issue 37

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Today's Topics:

   1. GFP again (Caroline Bass)
   2. paraffins (Steven Coakley)
   3. RE: GFP again (Favara, Cynthia (NIH/NIAID))
   4. Immunofluorescence about cartilage  (=?gb2312?q?=CC=EC=20=D0=C1?=)
   5. reply to murine CD4 and CD8 in brain sections (Gayle Callis)
   6. Re: paraffins (John Kiernan)
   7. GFP, the ongoing dilemma with DsRed included  (Gayle Callis)
   8. First time with frozen sections, long reply (Gayle Callis)
   9. Surgical Volume vs. number of tests (Patricia Karlisch)
  10. RE: Surgical Volume vs. number of tests (Bonner, Janet)
  11. Surgical Volume vs. number of tests  (Stephen Peters M.D.)
  12. NSH Self Assessment and Study Guide CD
      (Dick Paulson [Source Medical Products])
  13. coating slides for paraffin sections of CNS tissue (Julia Edgar)
  14. RE: need some help with frozen sections (C.M. van der Loos)
  15. immuno-LCM (Garry Ashton)
  16. RE: coating slides for paraffin sections of CNS tissue
      (Favara, Cynthia (NIH/NIAID))
  17. viability of placenta (Bernadette Weston)
  18. RE: Temp/Humidity Records? (Dawson, Glen)
  19. position (Dorothy.L.Webb@HealthPartners.Com)
  20. RE: coating slides for paraffin sections of CNS tissue
      (Due, Brice)
  21. RE: Temp/Humidity Records? (Willis, Donna)
  22. temp/humidity (Linda Blazek)
  23. Lyme Disease (Laurie Colbert)
  24. Re: Cleaning Acid (Long) (John A. Kiernan)


----------------------------------------------------------------------

Message: 1
Date: Mon, 25 Apr 2005 13:21:41 -0400
From: Caroline Bass 
Subject: [Histonet] GFP again
To: "
	"
	
Message-ID: <971ab99ff4f9d5a18b6bcaf122da48b6@bidmc.harvard.edu>
Content-Type: text/plain; charset=US-ASCII; format=flowed

Hey Guys,

I have been searching the internet and all sorts of forums, including 
histonet trying to find the answers to my GFP questions.  Essentially, 
I have a viral vector that I would like to inject into the liver which 
expresses EGFP.  I would like to have the easiest way to determine if 
it works or not.  I have heard that it is virtually impossible to 
detect native fluorescence with GFP, and that frozen cryostat sections 
completely lose the signal.  Am I missing something here?  There is a 
paper that is essentially doing the same thing that I would like to do, 
and they have enough signal that they can see it in the intact animal 
with a UV lamp, or in 6 micron cryosections without fixation or 
perfusion.  According to everything I read, this shouldn't work.  Does 
EGFP produce a stronger signal that can be seen in this way?

The paper is:  Nature Biotechnol 2005 Mar; 23(3):321-8.

Any suggestions on how to process the liver such that I can see the 
native EGFP signal would be greatly appreciated.  I have access to a 
cryostat and a microtome.  I can freeze the tissue, perfuse the mouse, 
whatever it takes.  I would prefer not to use immunostaining initially, 
as I have to section through entire lobes to visualize where the virus 
diffuses, etc.  I can use an antibody once I get a rough estimate of 
where the signal is, but we don't have enough money to immunostain 
every section.

Thanks,

Caroline Bass




------------------------------

Message: 2
Date: Thu, 21 Apr 2005 12:52:48 -0500
From: "Steven Coakley" 
Subject: [Histonet] paraffins
To: 
Message-ID: 
Content-Type: text/plain;	charset="us-ascii"



Does anyone have any experience with surgpath IF200 infiltration medium and
EM400 embedding medium.  I'm considering using 2 different paraffins instead
of one of the duel use paraffins.  Is infiltation really improved by using
the "softer" paraffin.  Any opinions recommended.  I'm starting up a new
histo lab.

Thanks,

Steven




------------------------------

Message: 3
Date: Mon, 25 Apr 2005 13:55:57 -0400
From: "Favara, Cynthia (NIH/NIAID)" 
Subject: RE: [Histonet] GFP again
To: 'Caroline Bass' ,
	"
	"
	
Message-ID:
	
Content-Type: text/plain

Caroline,

I am pretty much a novice at the GFP but have done some work on this. I can
tell you that the protein is very soluble and does not survive standard
fixation with NBF when working with frozen sections. There is a nice paper J
Histochem Cytochem. 2003 Mar;51(3):401-4 that discusses this and proposes a
vapor fixation. 

We have had good luck with formalin fixed paraffin embedded tissue using
anti GFP antibodies. In my hands there is considerable variation in the
sensitivity and signal /noise ratio. My suspicion is that due to the
solubility of the protein perfused specimens may be preferable. But I have
not tested this. 

We have started a project with grafting of GFP expressing tissue into a non
GFP expressing mouse and I can tell you that seeing the GFP in the fresh
tissue is not as obvious as some of the literature would lead one to
believe.

I am still working on this so I look forward to replies from others more
knowledgeable and experienced than I.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Caroline Bass [mailto:cbass@bidmc.harvard.edu] 
Sent: Monday, April 25, 2005 10:22 AM
To:  
Subject: [Histonet] GFP again

Hey Guys,

I have been searching the internet and all sorts of forums, including 
histonet trying to find the answers to my GFP questions.  Essentially, 
I have a viral vector that I would like to inject into the liver which 
expresses EGFP.  I would like to have the easiest way to determine if 
it works or not.  I have heard that it is virtually impossible to 
detect native fluorescence with GFP, and that frozen cryostat sections 
completely lose the signal.  Am I missing something here?  There is a 
paper that is essentially doing the same thing that I would like to do, 
and they have enough signal that they can see it in the intact animal 
with a UV lamp, or in 6 micron cryosections without fixation or 
perfusion.  According to everything I read, this shouldn't work.  Does 
EGFP produce a stronger signal that can be seen in this way?

The paper is:  Nature Biotechnol 2005 Mar; 23(3):321-8.

Any suggestions on how to process the liver such that I can see the 
native EGFP signal would be greatly appreciated.  I have access to a 
cryostat and a microtome.  I can freeze the tissue, perfuse the mouse, 
whatever it takes.  I would prefer not to use immunostaining initially, 
as I have to section through entire lobes to visualize where the virus 
diffuses, etc.  I can use an antibody once I get a rough estimate of 
where the signal is, but we don't have enough money to immunostain 
every section.

Thanks,

Caroline Bass


_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

Message: 4
Date: Tue, 26 Apr 2005 01:58:05 +0800 (CST)
From: =?gb2312?q?=CC=EC=20=D0=C1?= 
Subject: [Histonet] Immunofluorescence about cartilage 
To: Histonet@pathology.swmed.edu
Message-ID: <20050425175805.63358.qmail@web15506.mail.cnb.yahoo.com>
Content-Type: text/plain; charset=gb2312

Hello,all,
 
I am doing double immunofluorescence in bone sections. One protein was
mainly expressed in cytoplasm, but we found that some positive expressions
appeared among chondrocytes, moreover their expressions were stronger, my
teacher told me, it could be due to this condition that part cartilages were
broken during the staining so that this protein came out from chondrocytes,
then positive expressions were present among chondrocytes. If it is true,
what should I do to avoid this situation? If you have some experience about
it, please do me a favor! Thank you!
 
In addition, which methods can be used to reduce background? 
 
Guofeng Qian
 
 



---------------------------------
Do You Yahoo!?
注册世界一流品质的雅虎免=D1电邮

------------------------------

Message: 5
Date: Mon, 25 Apr 2005 12:16:02 -0600
From: Gayle Callis 
Subject: [Histonet] reply to murine CD4 and CD8 in brain sections
To: Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050425120037.01b0cba8@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Christophe Meiers wrote:

    Could  someone  please  give  advice regarding cd4 and cd8 staining of
    brain   tissue.    We  dissected  out  the  brain  of  a  mouse,  made
    longitudinal  section  midbrain,  one  half of the brain was placed in
    formalin for routine H & E, the other half was snap frozen for the cd4
    and  cd8.   The sections came out absolutely horrible with what looked
    like  exploded  tissue.  Should  the  brain  be treated in a different
    fashion ?  Would formalin interfere with staining CD4 and CD8 ?

*****You will have to frozen sections on fresh, unfixed snap frozen brain. 
Brain should be embedded in OCT, then it must MUST be snap frozen at very=20
cold temps with either a dry ice/2 methylbutane mixture or another adequate 
method.  Snap freezing has been discussed many times on Histonet, for 
methods do a search of Histonet Archives at www.histosearch.org.   Formalin 
totally kills murine CD4 and CD8 antigen, and no retrieval or enzyme 
digestion will ever recover the antigens. Make sure block temperature and=20
knife temperature are the same, equilibrate block (if stored at -80C) 20 
min or so before cutting.  Too cold a block means crunchy sections.

Cryosection brain at -16C, 5 um sections, pick up on Plus Charge slides, 
air dry overnight, fix in 75%acetone/25% absolute ethanol (no substitute on 
alcohol can be used) for 5 min at room temperature, go directly to a buffer 
rinse FROM the fixative, 3 changes of buffer, and proceed with staining. Do 
not store your frozen sections in cryostat after cutting, go directly to 
air drying.  We put our sections over a 16 mesh silica gel to ensure 
dryness.   Some people use acetone fixation at 4C for 10 min, but the 
acetone/alcohol improves morphology and keeps excellent immunostaining.

Peroxidase block, DAKO S2001 for 10 min

Normal serum block for 30 min (10% goat/2.5%mouse in TBS or Dulbeccos PBS=20
with 0.05% Tween 20.  We add 0.2% goat serum to rinse buffers along with 
0.05% Tween 20

Avidin/biotin block kit Vector

CD4 Rat antimouse BD Pharmigen 1:500 (0.5mg/ml conc) in 5% goat serum.
CD8 Rat antimouse 1:200 (0.5mg/ml conc) in 5% goat serum
IgG isotype matched controls for these antibodies at same concentration in 
ug/ml is negative control
Incubate 30 min, RT

Goat antiRat F(ab')2 frag of IgG (0.5mg.ml) diluted 1:250 in normal serum=20
block (yes! with mouse serum) for 30 min

Strepavidin-HRP 1:500 from Biosource/TAGO diluted in rinse buffer, 20 min

AEC+ DAKO control color development of positive control (normal spleen 
works) for approx 3 min

Rinse, counterstain, and mount with aqueous mounting media.






Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)





------------------------------

Message: 6
Date: Mon, 25 Apr 2005 14:33:16 -0400
From: John Kiernan 
Subject: Re: [Histonet] paraffins
To: steven.coakley@mirusbio.com
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <426D37EC.6949BC0E@uwo.ca>
Content-Type: text/plain; charset=us-ascii

The subject of different paraffins was discussed at some
length on Histonet more than 5 years ago. The most 
authoritative comments seemed to be from Russ Allison.
His principal conclusion was that ingredients other
than the hydrocarbons (the wax itself) made no difference
to cutting properties. Russ has published in this field;
the following list is probably incomplete.

Allison RT (1978) The crystaline nature of histology waxes: 
a preliminary communication. Medical Laboratory Sciences 
35: 355-363.

Allison RT (1979) The crystaline nature of histology waxes: 
the effects of microtomy on the micro-structure of paraffin 
wax in sections. Medical Laboratory Sciences 36: 359-372.

Allison RT, Lloyd D (1996) Measuring infiltration during 
paraffin wax processing for histology. British Journal of 
Biomedical Science 53: 235-237.

Allison RT, Bryant D (1998) Effects of processing at 45C on 
staining. Biotechnic & Histochemistry 73: 128-136.

Allison RT (2002) Tissue processing and sectioning. Chapter
7 in  Microscopy and Histology for Molecular Biologists
(Kiernan JA & Mason I eds). London: Portland Press. 
pp 144-169.

In the most recent of these publications, Russ concedes that 
there may be some circumstances where addition of DMSO or a 
synthetic polymer could be advantageous. Unfortunately the
archives at www.histosearch.com do not include the older
correspondence about waxes. Russ Allison does not seem to
be around on Histonet these days; a pity!
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Steven Coakley wrote:
> 
> Does anyone have any experience with surgpath IF200 infiltration medium
and
> EM400 embedding medium.  I'm considering using 2 different paraffins
instead
> of one of the duel use paraffins.  Is infiltation really improved by using
> the "softer" paraffin.  Any opinions recommended.  I'm starting up a new
> histo lab.
> 
> Thanks,
> 
> Steven
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

Message: 7
Date: Mon, 25 Apr 2005 12:42:39 -0600
From: Gayle Callis 
Subject: [Histonet] GFP, the ongoing dilemma with DsRed included 
To: Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050425121642.01b40a58@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Dear Caroline,

You wrote:
I have been searching the internet and all sorts of forums, including
histonet trying to find the answers to my GFP questions.  Essentially,
I have a viral vector that I would like to inject into the liver which
expresses EGFP.  I would like to have the easiest way to determine if
it works or not.  I have heard that it is virtually impossible to
detect native fluorescence with GFP, and that frozen cryostat sections
completely lose the signal.  Am I missing something here?  There is a
paper that is essentially doing the same thing that I would like to do,
and they have enough signal that they can see it in the intact animal
with a UV lamp, or in 6 micron cryosections without fixation or
perfusion.  According to everything I read, this shouldn't work.  Does
EGFP produce a stronger signal that can be seen in this way?
The paper is:  Nature Biotechnol 2005 Mar; 23(3):321-8.
Any suggestions on how to process the liver such that I can see the
native EGFP signal would be greatly appreciated.  I have access to a
cryostat and a microtome.  I can freeze the tissue, perfuse the mouse,
whatever it takes.  I would prefer not to use immunostaining initially,
as I have to section through entire lobes to visualize where the virus
diffuses, etc.  I can use an antibody once I get a rough estimate of
where the signal is, but we don't have enough money to immunostain
every section.

***We recently undertook a similar project with undecalcified murine nasal 
turbinate and small intestine tissue sections from mouse.

In order to do immunofluorescence staining for a CD marker or dendritic 
cell, we were required to fix with acetone or acetone/alcohol, which 
immediately killed the eGFP and it failed to glow. This was also going be a 
problem with DsRED, a bioluminescent protein from Coral rather than
jellyfish.

One major problem is increasing autofluorescence in tissues by using 
formalin or paraformaldehyde fixatives. We could never get the best of both 
worlds with eGFP or DsRed.

Final solution to the problem:

With DsRed, unfixed fresh snap frozen tissues were cryosectioned at 5 um,=20
mounted on Erie Plus Gold slides (a plus charged surface for extra holding 
power),and air dried for 30 min or more in front of a fan at RT.  Air dried 
sections were rinsed one slide at time to prevent unfixed section loss with 
2 changes PBS gentle agitation.  We wanted to get rid of OCT. A coverslip=20
was mounted with Prolong Gold antifade mounting media, ready to use 
containing DAPI for DNA/RNA.

Results:  spectacular DsRed where it was supposed to be, with nuclei 
counterfluorescing a lovely blue from DAPI staining DNA/RNA.  The mounting 
media is a hard set.  The only autofluorescence is natural, but does NOT 
interfere with the brighter signal of DsRed, and would not interfere with=20
eGFP either.

We are going to do this with eGFP next week to have green fluorescence with 
the DAPI stained nuclei.

I have a review of autofluorescence I am going to attach to you.  For more 
on eGFP and DsRed, go to Clontech website and download their Living Colours 
Manuals for these proteins.

When you get to immunostaining, say for for a CD marker along with 
eGFP,  we did our acetone/alcohol fixation, but detected eGFP with Goat 
antiGFP from Rockland (a superb antibody that is NOT expensive!) and came=20
back with Donkey antiGoat-FITC, along with a biotinylated CD marker 
antibody and Strepavidin Alexa 555.  This worked well for double 
fluorescence with the CD marker.  As for DsRed, the antiDsRed antibody is=20
pricey, so doing IFA staining for GFP has been more cost effective.








------------------------------

Message: 8
Date: Mon, 25 Apr 2005 13:02:41 -0600
From: Gayle Callis 
Subject: [Histonet] First time with frozen sections, long reply
To: Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050425124550.01b4c780@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

You wrote:
I am starting to stain frozen sections for the first time. Until now I
have only stained paraffin embedded sections and so was hoping for some
pointers.I contacted our histopathology lab who provided me with the
following
information: They don't use any antigen retrieval method.
They pretreat the slides only with methanol for 10min followed by drying
before staining. Then they proceed with the normal steps of staining and 
dehydration.
I have however read that acetone can be used instead of the methanol.
What is the difference that this provides? I am trying to stain for Stat5=2E
Is there anything els I should keep in mind when handling frozen sections?

Questions:

human tissue, animal tissue? Is the tissue fresh, unfixed or prefixed prior 
to snap freezing?

Have you snap frozen (needed to minimize freezing artifact from water ice=20
crystal formation) the tissues rather than cryostat freezing (too warm)?

What antigens do you want to stain for?

Fixation is dependent on the antigen, some will be fine with acetone, some 
may work better with another fixative.

Antigen retrieval on frozen sections of fresh unfixed tissues is not needed 
as long a formalin or even paraformaldehyde is avoided.  In general, AR 
tends to damage FS.

If you plan to do CD marker staining, methanol can cause poor staining of=20
these due to methanolic bridge formation.  Avoid methanol as a fixative or 
in peroxidase block, use DAKO S2002 peroxidase block designed for gentle 
endog perox blocking, it doesn't contain MEOH and wil not chew section off 
the slide.  Hydrogen peroxide can be put into PBS instead.

Antibodies also need to be optimized since FS permit lower conc of 
antibodies.   Dilution panels should be done.

What does the spec sheet or manufacturer recommend for fixation of FS for=20
your STAT5 antibody?  You can always call their tech services and ask.

Be gentle, FS are more fragile, and air dry them AFTER sectioning, DO NOT=20
STORE newly cut setions in a cryostat. Avoid water condensation and freeze 
thawing of sections - this can damage antigens.   Air drying overnight in=20
front of a fan, or over dessicant is a good way to insure properly dried 
sections.

Check Histonet Archives www.histosearch.org, it has tons of info on HOW to 
do frozen sections, fixation, blocking, and staining. Chris van der Loos 
has provided many excellent replies on frozen section IHC. DakoCytomation=20
website has Immunochemical Staining Methods Handbook, 3rd Edition with tons 
of hints on what you want to do.

Good luck





Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)





------------------------------

Message: 9
Date: Mon, 25 Apr 2005 17:49:21 -0400
From: "Patricia Karlisch" 
Subject: [Histonet] Surgical Volume vs. number of tests
To: 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Histonetters:
  First, thank you all for the wonderful responses that you sent
regarding the immunohistochemical staining relative to the volume of
stains done per day.  Your responses were very helpful.  I would like to
pose another question to you.  Do you think there is over utilization
with ordering immunohistochemistries on a daily basis?  Relative to your
surgical cases what would you say that your percentage (or actual
numbers) of immunohistochemical cases  are and how many slides would
that equate to.
  For example:  6-8% of all our cases require immunohistochemistry,
thus as volume increases so do our immuno stains.  We do on average 1400
immuno slides/month which includes surgical, biopsy, bone marrow,
Cytology FNA's and skins with one tech on rotation and two stainers with
a capacity of 40 slides total. (Our yearly surgicals are about 34000
cases). We do very little research in this lab and minimal duplicate
staining for file, study or education.    
  Do any of you employ a method to discourage over utilization of
immuno staining?
  Thank you again for any advice you can give me.
   Pat Karlisch

Pat Karlisch
Supervisor, Histology, Pathology and Laboratory Medicine
Penn State Milton S. Hershey Medical Center
Mail Code H179
Hershey, PA 17033
Phone (717) 531-6072
Fax: (717) 531- 7741
email: pkarlisch@psu.edu




------------------------------

Message: 10
Date: Mon, 25 Apr 2005 18:18:45 -0400
From: "Bonner, Janet" 
Subject: RE: [Histonet] Surgical Volume vs. number of tests
To: "'Patricia Karlisch'" ,
	histonet@lists.utsouthwestern.edu
Message-ID: <07AB60D5D7B9754EBF56F360F98D083DEB424D@fh2k093.fhmis.net>
Content-Type: text/plain;	charset="iso-8859-1"

 The workload/slides seems in line with ours.  The only discouraging factor
to ordering certain immunos is the ability to charge the patient/insurance
company AND to justify the order/charges if called on the carpet.

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Patricia
Karlisch
Sent: Monday, April 25, 2005 5:49 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Surgical Volume vs. number of tests


Histonetters:
  First, thank you all for the wonderful responses that you sent
regarding the immunohistochemical staining relative to the volume of
stains done per day.  Your responses were very helpful.  I would like to
pose another question to you.  Do you think there is over utilization
with ordering immunohistochemistries on a daily basis?  Relative to your
surgical cases what would you say that your percentage (or actual
numbers) of immunohistochemical cases  are and how many slides would
that equate to.
  For example:  6-8% of all our cases require immunohistochemistry,
thus as volume increases so do our immuno stains.  We do on average 1400
immuno slides/month which includes surgical, biopsy, bone marrow,
Cytology FNA's and skins with one tech on rotation and two stainers with
a capacity of 40 slides total. (Our yearly surgicals are about 34000
cases). We do very little research in this lab and minimal duplicate
staining for file, study or education.    
  Do any of you employ a method to discourage over utilization of
immuno staining?
  Thank you again for any advice you can give me.
   Pat Karlisch

Pat Karlisch
Supervisor, Histology, Pathology and Laboratory Medicine
Penn State Milton S. Hershey Medical Center
Mail Code H179
Hershey, PA 17033
Phone (717) 531-6072
Fax: (717) 531- 7741
email: pkarlisch@psu.edu


_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

Message: 11
Date: Mon, 25 Apr 2005 18:24:33 -0700 (PDT)
From: "Stephen Peters M.D." 
Subject: [Histonet] Surgical Volume vs. number of tests 
To: Histonet@lists.utsouthwestern.edu
Message-ID: <20050426012433.21115.qmail@web30410.mail.mud.yahoo.com>
Content-Type: text/plain; charset=us-ascii

Dear Pat, 
 I will offer a pathologists point of view. The actual percent cases which
will need immuno
 will vary depending on several factors. 
First the case mix. You are in an academic center. I would expect you would
have a 
larger percentage  of demanding cases than a small private hospital. For
example if you 
did only hernias, gallbladders and seb kers, you would not need any immunos.
If you are 
doing all lymphomas and  and soft tissue tumors you would have a %100 immuno
 rate. 
Experience and degree of specialization of the pathologists will play a
role.
 In many situations the less experienced people tend to order a bit more
than the more 
experienced people because of the lack of confidence that comes  with
experience. This is a natural evolution of the life of the pathologist much
as your experience has tought you to find the simplest path.
More academic pathologists with subspecialties may study certain cases in
more 
depth because of their fields of interest. 
Also being an academic center with residents pathologists will often order
more 
complete panals even though the diagnosis is clear for the benifit of the
residents
 experience.
 Taking those facts out of the equation, I would be surprised if everyone
reading this has 
not experienced increasing percentages of immuno requests over the past
years.
 The number of antibodies required to carry out what would be considered 
" standard of practice" by our friends in the legal profession has grown
enormously.  And 
they will continue to grow. They are extremely helpful and represent one of
the most 
significant advances in our profession. But have no fear. I predict the day
is coming when 
your percentage of immunos will start to creep down only to be replaced by
more 
specific, more intellectually demanding, and probably more expensive
molecular methods!  
 
As far as trying to discourage over utilization, that is a budget vs.
medical care  issue and
 is under the perview of your medical director. It is his job to optimize
care 
while keeping to the budget. You  would have to be an experienced surgical
pathologist
 to even begin make this judgement. One thing you could do to see if there
is a particular culprit is to audit the ordering patterns of the different
pathologists. Your computer system may be able to give you this easily.If
you believe somebody is being wasteful with ordering
 of studies it is your job to bring it up with your superiors. When it works
its way up, it can 
be looked at by a someone who can evaluate the situation from both sides.=20
If there is an undo amout of tests being ordered this could possibly be
reduced by 
more intradepartmental consultation. Sometimes sharing cases before immunos
are 
ordered can prevent over ordering. It can also backfire and end up getting
you more requests! When I show one of my cases to colleagues invariably I
end up ordering a few more stains to 
satisfy their curiosity.
If the hospital is pressuring you from the budget side then take a good hard
look at where you
can save without jeopardizing patient care. I would rather them take away
the heat and the toilet paper than force me take guesses by cutting back on
necessary studies. 
 


Stephen Peters M.D. 
Vice Chairman of Pathology
Hackensack University Medical Center 
201 996 4836
 
Pathology Innovations, LLC 
410 Old Mill Lane, 
Wyckoff, NJ 07481 
201 847 7600 
www.pathologyinnovations.com 






------------------------------

Message: 12
Date: Tue, 26 Apr 2005 02:24:19 -0500
From: "Dick Paulson [Source Medical Products]"
	
Subject: [Histonet] NSH Self Assessment and Study Guide CD
To: 
Message-ID:
	

	
Content-Type: text/plain;	charset="us-ascii"

For those of you who ordered the CD but couldn't get it to work.. Good news.

NSH sent me a CD to test. This is my response.

The CD works on all the systems I tested.

I tested on the following operating systems:
Windows 98
Windows 2000
Windows XP Home Edition
Windows XP Professional Edition

All the operating systems have the most up to date Service Packs and updates
applied.

I tested with the disk in the CD drive and also created a folder and copied
the contents of the CD to a folder I created on disk.  I also created a
folder on a Windows 2000 server with the CD contents and ran the program
from a mapped drive to the server.

I went through 2 Studies and 1 Test to completion.

Everything works.  This is a great learning tool.

I hope this helps.

The CD was sent on the 19th but I just got it today (25th).

If you have any questions, please do not hesitate to contact me directly.

Thank you,

Source Medical Products
Dick Paulson
dpconsult@earthlink.net
Phone   (800) 393-6345 





------------------------------

Message: 13
Date: Tue, 26 Apr 2005 09:50:05 +0100
From: "Julia Edgar" 
Subject: [Histonet] coating slides for paraffin sections of CNS tissue
To: 
Message-ID: <000901c54a3c$f17e3300$cdead182@vet.gla.ac.uk>
Content-Type: text/plain;	charset="iso-8859-1"

Dear All
We've been using APES coated slides for collecting paraffin embedded CNS
tissue for immunohistochemistry (with microwave treatment) but the sections
are becoming unstuck, to some extent, during processing. I will be grateful
if you can tell me what you are coating slides with for small pieces of CNS
tissue.
Thank you
Julia
Julia Edgar BSc (Hons), PhD
University of Glasgow
Tel: 0141 330 5818
e-mail: je22r@udcf.gla.ac.uk




------------------------------

Message: 14
Date: Tue, 26 Apr 2005 11:01:54 +0200
From: "C.M. van der Loos" 
Subject: [Histonet] RE: need some help with frozen sections
To: histonet@lists.utsouthwestern.edu
Message-ID: <42f77b432f60.432f6042f77b@amc.uva.nl>
Content-Type: text/plain; charset="us-ascii"


   Eva,

   Some DO's and DON'T's with immunostaining of cryostat tissue sections:
     * After  cutting, let the sections dry overnight at room temperature
       under a ventilator
     * Fix  with  cold  acetone (10 min, 4C). Some people apply a "double
       acetone   fixation"  method:  2x  10  min  acetone  fixation  with
       air-drying in between. This may improve the tissue morphology.
     * The  quality  of the acetone should be P.A. grade and don't use it
       twice. Re-distilled acetone cannot be used for fixation of cryo's.
       Traces of water in the acetone ruins the tissue morphology.
     * Be  aware  that  acetone  is not a real fixative like NBF. Acetone
       just  solves  the  fatty  membranes  and  coagulate  the proteins.
       Cryostat  tissue  sections remain quite vulnerable with respect to
       surfactants  like Triton-X100,  Tween-20. These should be avoided.
       Be  aware  that in some autostainer washbuffers surfactants may be
       included.
     * To  solve  this  problem of tissue vulnerability an extra fixation
       (after  acetone-fixation  and  air-drying)  with Zamboni's (1 min,
       RT) is optional. This extra fixation step also improves the tissue
       morphology.  However,  depending on the antigen to be IHC stained,
       this step may also decrease the IHC staining intensity.
     * The  application  of methanol, either as a fixative or as base for
       endogenous   peroxidase   activity  blocking  is (at  least to  my
       vision) absolutely  excluded.  For  example, most human CD markers
       are completely destroyed by methanol. On the other hand I am aware
       of investigators  who  are  using successfully an acetone/methanol
       mixture for fixation of mouse cryo's for staining CD4, CD8 etc.
     * Acetone   is   not   a   good   fixative   when  staining  nuclear
       antigens. This  fixation will  lead to quite fuzzy stained nuclei.
       Instead,  a  NBF-fixation  (5  min,  RT)  works out well. Although
       NBF-fixation is used: do NOT apply heat-induced antigen retrieval.
     * Some  antigens  associated  with  fatty  structures  (for example,
       oxLDL,  or apoB) will solve into acetone. In this case NBF (5 min)
       can be tested. Again, NO antigen retrieval!
     * Endogenous peroxidase activity can be blocked with 0.1% Na-azide +
       0.3%  peroxide in PBS or TBS (20 min, RT). This kills at least the
       endogenous peroxidase activity in erythrocytes effectively, but in
       neutrophils some will remain. If endogenous peroxidase activity of
       neutrophils  is  a  real  problem, glucose oxidase blocking method
       is optional (see Histonet archives).
     * At the end of the IHC staining procedure, after the chromogen step
       wash your slides with tap water. NEVER use distilled water as this
       will ruin the tissue section completely!!!!
     * Unlike  with  FFPE  sections,  aqueous solutions (depending on the
       chromogen  used  of  course)  is  no problem for mounting cryostat
       tissue sections.
     * The  concept  of  first cutting a tissue section and than fixation
       means  "post-fixation".  This  is  something different from a FFPE
       section that  is  first  fixed  as  a block, than embedded and cut
       ("pre-fixation").  This  means that soluble antigens may leak away
       from  a "post-fixed"  tissue  section. Be  aware  of this when IHC
       staining  any small protein (<50KD), cytokine, chemokine, hormone,
       etc.

   I hope this isn't too much frightening you. Happy staining!

   Chris van der Loos, PhD
   Dept. of Pathology
   Academical Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389
   e-mail: [1]c.m.vanderloos@amc.uva.nl




   ----- Original Message -----
                                            From 
   Eva C Anderson 
                                            Date 
   Mon, 25 Apr 2005 09:59:34 -0400
                                              To 
   histonet@lists.utsouthwestern.edu
                                         Subject 
   [Histonet] need some help with frozen sections

   Good morning,
   I am starting to stain frozen sections for the first time. Until now I
   have  only  stained  paraffin  embedded sections and so was hoping for
   some
   pointers.
   I contacted our histopathology lab who provided me with the following
   information:
   They don't use any antigen retrieval method.
   They  pretreat  the  slides  only  with methanol for 10min followed by
   drying
   before staining.
   Then they proceed with the normal steps of staining and dehydration.
   I have however read that acetone can be used instead of the methanol.
   What  is  the  difference that this provides? I am trying to stain for
   Stat5.
   Is  there  anything  els  I  should  keep in mind when handling frozen
   sections?
   Would  be  very  grateful  for  any information you could provide this
   rookie
   with.
   Thank you,
   Eva

References

   1. mailto:c.m.vanderloos@amc.uva.nl


------------------------------

Message: 15
Date: Tue, 26 Apr 2005 11:14:18 +0100
From: "Garry Ashton" 
Subject: [Histonet] immuno-LCM
To: 
Message-ID:
	
Content-Type: text/plain;	charset="us-ascii"

Dear all,
I'm currently trying to re-optimize an immuno technique on frozen
sections that allows for the production of quality RNA after laser
capture.
Presently I have found that not allowing the sections to dry, storing at
-80, and using a much shortened immuno method (2 mins max in all ab
incubations), plus a shortened colour reaction, with an RNase inhibitor
added at the ab and colour steps gives me reasonable results.
Does anybody else in histoland use this technique regularly, and if so
have they found results similar to me.
Many thanks.
Garry
 
 
 
PICR
UK
 
--------------------------------------------------------

 
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('the intended recipient') to whom it was addressed. Any views or opinions
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within the meaning of applicable law. Accordingly any dissemination,
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by any person other than the intended recipient may constitute a breach of
civil or criminal law and is strictly prohibited. If you are NOT the
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------------------------------

Message: 16
Date: Tue, 26 Apr 2005 09:05:54 -0400
From: "Favara, Cynthia (NIH/NIAID)" 
Subject: RE: [Histonet] coating slides for paraffin sections of CNS
	tissue
To: 'Julia Edgar' ,
	histonet@lists.utsouthwestern.edu
Message-ID:
	
Content-Type: text/plain

Julia,

I work with primarily rodent CNS tissue and use Superfrost/Plus I get them
from Fischer in the US Cat# 22-034-979 and very rarely have any tissue loss
generally when I neglect to properly dry the slide. Hope this is helpful.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

Disclaimer: 
The information in this e-mail and any of its attachments is confidential
and may contain sensitive information. It should not be used by anyone who
is not the original intended recipient. If you have received this e-mail in
error please inform the sender and delete it from your mailbox or any other
storage devices. National Institute of Allergy and Infectious Diseases shall
not accept liability for any statements made that are sender's own and not
expressly made on behalf of the NIAID by one of its representatives

-----Original Message-----
From: Julia Edgar [mailto:je22r@udcf.gla.ac.uk] 
Sent: Tuesday, April 26, 2005 1:50 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] coating slides for paraffin sections of CNS tissue

Dear All
We've been using APES coated slides for collecting paraffin embedded CNS
tissue for immunohistochemistry (with microwave treatment) but the sections
are becoming unstuck, to some extent, during processing. I will be grateful
if you can tell me what you are coating slides with for small pieces of CNS
tissue.
Thank you
Julia
Julia Edgar BSc (Hons), PhD
University of Glasgow
Tel: 0141 330 5818
e-mail: je22r@udcf.gla.ac.uk


_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

Message: 17
Date: Tue, 26 Apr 2005 09:17:11 -0400
From: "Bernadette Weston" 
Subject: [Histonet] viability of placenta
To: histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: text/plain; format=flowed

How long can a placenta be refrigerated and still be viable for testing as 
well as Histology?

Bernadette Weston HT
Histology Supervisor
Barberton Citizens Hospital
Barberton, OH





------------------------------

Message: 18
Date: Tue, 26 Apr 2005 08:16:38 -0500
From: "Dawson, Glen" 
Subject: RE: [Histonet] Temp/Humidity Records?
Cc: histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: text/plain;	charset="iso-8859-1"


Just got back from vacation but I feel the need to comment on this.
Although I haven't heard of this one, it would not surprise me.
Documentation, documentation, documentation; it is the only thing between us
and utter chaos...give me a break.

Each day, my staff and I do our IHC runs that require heat.  My instructions
are very straight forward; unless the thermometer reads at least 97 degrees,
the run cannot continue.  If the bath isn't up to temp, the slides wait
until it is.  We check and check and check this same thermometer many times
a day and the heat runs don't continue until the magic number is reached.
Still, every day, we must lug the temperature log out and put in that entry.
Maybe this is a bad example but I don't see how writing an entry in a
temperature log for a reading we all know by heart is keeping the walls of
this lab from crumbling down.

At some point, doesn't the additional, meaningless documentations added onto
the CAP inspection roster become just that, meaningless.  I picture a person
somewhere in a dark room racking his or her brain 24/7 for more "required"
documentation for CAP inspections because every year I think that CAP
couldn't possibly come up with more things to document, they do.  

I'm rambling, long story short, I'm sick of all this documentation.

My Opinion,

Glen Dawson  BS, HT & QIHC (ASCP)
IHC Manager
Milwaukee, WI



------------------------------

Message: 19
Date: Tue, 26 Apr 2005 09:18:08 -0500
From: Dorothy.L.Webb@HealthPartners.Com
Subject: [Histonet] position
To: histonet@lists.utsouthwestern.edu
Message-ID:
	
Content-Type: text/plain


We have a full time opening for a histotechnician, day shift, and on call
every 7th Saturday between the hours of  0800 and 1430.  This position
rotates into all aspects of histology and will be trained to be a "back-up"
in our IHC area.  We are a large city trauma 1 hospital in St. Paul, MN.
with 12 pathologists on board.  A nice place to work, so, come on board!!
You can contact me by Email if interested or go on line to regions.com for
an application site and full job description!


________________________________________

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are addressed. If you are not the intended recipient or the individual
responsible for delivering the e-mail to the intended recipient, please
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use, dissemination, forwarding, printing, or copying of this e-mail
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If you have received this e-mail in error, please immediately notify
the HealthPartners Support Center by telephone at (952) 967-6600.
You will be reimbursed for reasonable costs incurred in notifying us.

------------------------------

Message: 20
Date: Tue, 26 Apr 2005 10:49:03 -0400
From: "Due, Brice" 
Subject: RE: [Histonet] coating slides for paraffin sections of CNS
	tissue
To: "Favara, Cynthia \(NIH/NIAID\)" ,	"Julia
	Edgar" , 
Message-ID:
	<59C772E2D8EDF345AE1601F5B60B3CB50279CA@PHSXMB7.partners.org>
Content-Type: text/plain;	charset="iso-8859-1"

Hello Julia, I work in a clinical neuropathology lab. We do impox, silver
stains, etc. on both charged (superfrost) and uncharged slides. We also do
giant
sctions up to 4in. x 6 in. on plain (uncharged) glass slides. We rarely have
adhesion problems. You must make absolutely sure the sections are fully
dried
before you melt and deparaffinize them. If you melt the paraffin too soon, a
water film will become trapped and the sections will begin lifting in
xylene.
The best way is to leave the slides in a 37C oven overnight. Second best is
room
temp overnight. In my experience, the second most important factor in
adhesion
is section flatness. Wrinkles will not only fill up with water, but they
will
also not make contact with the slide. In some autopsy cases we also use
STA-ON
(chromated gelatin) adhesive, either in the water bath, or smeared on the
slide,
but the drying rules above are still mandatory. If overnight is too long for
you, first try it, and then try cutting back the drying times until your
problems reappear.

Good luck,
-brice
Neuropathology Lab
Brigham & Women's Hospital
Boston

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Favara,
Cynthia (NIH/NIAID)
Sent: Tuesday, April 26, 2005 9:06 AM
To: 'Julia Edgar'; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] coating slides for paraffin sections of CNS
tissue


Julia,

I work with primarily rodent CNS tissue and use Superfrost/Plus I get them
from Fischer in the US Cat# 22-034-979 and very rarely have any tissue loss
generally when I neglect to properly dry the slide. Hope this is helpful.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

Disclaimer: 
The information in this e-mail and any of its attachments is confidential
and may contain sensitive information. It should not be used by anyone who
is not the original intended recipient. If you have received this e-mail in
error please inform the sender and delete it from your mailbox or any other
storage devices. National Institute of Allergy and Infectious Diseases shall
not accept liability for any statements made that are sender's own and not
expressly made on behalf of the NIAID by one of its representatives

-----Original Message-----
From: Julia Edgar [mailto:je22r@udcf.gla.ac.uk] 
Sent: Tuesday, April 26, 2005 1:50 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] coating slides for paraffin sections of CNS tissue

Dear All
We've been using APES coated slides for collecting paraffin embedded CNS
tissue for immunohistochemistry (with microwave treatment) but the sections
are becoming unstuck, to some extent, during processing. I will be grateful
if you can tell me what you are coating slides with for small pieces of CNS
tissue.
Thank you
Julia
Julia Edgar BSc (Hons), PhD
University of Glasgow
Tel: 0141 330 5818
e-mail: je22r@udcf.gla.ac.uk


_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet



------------------------------

Message: 21
Date: Tue, 26 Apr 2005 09:55:20 -0500
From: "Willis, Donna" 
Subject: RE: [Histonet] Temp/Humidity Records?
To: "Dawson, Glen" 
Cc: histonet@lists.utsouthwestern.edu
Message-ID:
	<4483B6EE0FA82A4EAED2B1E161E5E41147C7BD@ftwex02.txhealth.org>
Content-Type: text/plain;	charset="us-ascii"

Glen,
Just wait until the CLSI (formally NCCLS) Microwave Device Use in the
Histolog Laboratory Guideline is added to CAP.

Donna Willis
Histology Lab Manager
Harris Methodist Fort Worth, Tx

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Dawson,
Glen
Sent: Tuesday, April 26, 2005 8:17 AM
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Temp/Humidity Records?



Just got back from vacation but I feel the need to comment on this.
Although I haven't heard of this one, it would not surprise me.
Documentation, documentation, documentation; it is the only thing
between us and utter chaos...give me a break.

Each day, my staff and I do our IHC runs that require heat.  My
instructions are very straight forward; unless the thermometer reads at
least 97 degrees, the run cannot continue.  If the bath isn't up to
temp, the slides wait until it is.  We check and check and check this
same thermometer many times a day and the heat runs don't continue until
the magic number is reached. Still, every day, we must lug the
temperature log out and put in that entry. Maybe this is a bad example
but I don't see how writing an entry in a temperature log for a reading
we all know by heart is keeping the walls of this lab from crumbling
down.

At some point, doesn't the additional, meaningless documentations added
onto the CAP inspection roster become just that, meaningless.  I picture
a person somewhere in a dark room racking his or her brain 24/7 for more
"required" documentation for CAP inspections because every year I think
that CAP couldn't possibly come up with more things to document, they
do.  

I'm rambling, long story short, I'm sick of all this documentation.

My Opinion,

Glen Dawson  BS, HT & QIHC (ASCP)
IHC Manager
Milwaukee, WI

_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet




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only for the use of the individual or entity to which it is addressed, and
may contain information that is PRIVILEGED, CONFIDENTIAL, and exempt from
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------------------------------

Message: 22
Date: Tue, 26 Apr 2005 11:32:28 -0400
From: "Linda Blazek" 
Subject: [Histonet] temp/humidity
To: histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Where in the CAP guidelines does it say that humidity must be documented?
Linda
 
Linda  Blazek, HT (ASCP)
Department of Pathology
Children's Medical Center
Dayton, Ohio  45404
(937) 641-3358
fax (937)641-5482
blazekl@childrensdayton.org




------------------------------

Message: 23
Date: Tue, 26 Apr 2005 09:02:22 -0700
From: "Laurie Colbert" 
Subject: [Histonet] Lyme Disease
To: 
Message-ID:
	
<0BE6ADFAE4E7E04496BF21ABD346628001C5C2DC@EXCHANGE1.huntingtonhospital.com>
	
Content-Type: text/plain;	charset="iso-8859-1"

Will a warthin starry diagnose lyme disease?  Is there an IHC stain that
will stain for this?

Laurie Colbert



------------------------------

Message: 24
Date: Tue, 26 Apr 2005 12:28:58 -0400
From: "John A. Kiernan" 
Subject: Re: [Histonet] Cleaning Acid (Long)
To: "Scott, Allison D" 
Cc: "'histonet@lists.utsouthwestern.edu'"
	
Message-ID: <426E6C4A.D7D3D0A0@uwo.ca>
Content-Type: text/plain; charset=us-ascii

An answer to a question asked last week about 
cleaning glassware for a silver staining method.
Skip to the bottom line if you don't want to
know the reasons.

Glassware that has been used for silver methods can
collect traces of metallic silver - sometimes enough
to see as a mirror or grey marks. If the glassware
is used again for silver staining, these traces serve
as nuclei for deposition of more silver, and they
are bigger than the desired nucleation sites in
the sections. The staining method may simply fail 
or the chemical reaction may go wild, with nonspecific
deposition of silver all over the place. 

Silver is soluble in nitric acid. My cleaning technique 
is to put a little concentrated HNO3 in the vessel
(Coplin jar or larger tank) and carefully move it over
all the inside surface, over a sink with running water
so that any spilled drops of acid are quickly diluted.
Visible silver (look in the corners) disappears 
instantly, so smaller amounts must also be removed.
Pour the used nitric acid into a beaker containing 
tap water (for later neutralization and disposal). If
the tap water becomes opalescent you have removed a
significant amount of silver from the glass.

Next - and this is important - Fill the vessel with 
PURE (eg distilled) water and empty it; do this twice 
so that the concentration of silver ions in the water
adhering to the sides is infinitessimal. Tap water 
must not be used for these washes because it always
contains anions (chloride, bicarbonate, others?) that
form insoluble silver salts. Any colloidal silver 
chloride particles that stay on the glass will be
partly reduced to silver by exposure to light and
can be expected to provide nucleation sites in
later silver staining methods. Finally dry the
glassware by letting it drain and store the vessels
upside-down in a closed cupboard.

Deposited silver is not the only kind of dirt that
can spoil silver staining. Any kind of organic 
chemical deposit (such as a fragment of a section)
or even residue from evaporated tap water will 
work in the same way. Concentrated nitric acid
quickly oxidizes and dissolves pretty well everything,
with one notable exception. 

The exception is metallic gold. This can replace 
deposited silver in glassware used for gold-toning, 
a procedure often used to improve contrast in
silvered preparations. Gold on glass may appear
only as a light purple discoloration. Any colour
that resists nitric acid is probably gold. It can be
removed with aqua regia, which is a 3:1 mixture of
concentrated nitric:hydrochloric acids. Make and
use aqua regia in a fume hood because it emits 
fumes of chlorine and nitrogen oxides. I have
resorted to aqua regia 3 or 4 times (in >30 years)
to get rid of gold on glass. The obvious way to
prevent contamination is to reserve certain jars
and dishes for gold-toning and nothing else. This 
is not very practical if we do many different
methods and do not have a cupboard big enough for 
all jars that might carry catalytic contaminants.

Some people use Farmer's reducer (a solution
containing potassium ferricyanide and sodium
thiosulphate). This is an altogether gentler
liquid than nitric acid and it can dissolve silver 
from black & white photographs. The action of
Farmer's reducer on visibly discoloured glass is 
very slow, and this mixture is not going to 
destroy insoluble organic forms of dirt such
as bits of tissue. 

Bottom line: Nitric acid, followed by pure (not
tap) water. 
 
John Kiernan
London, Canada.
________________________________
"Scott, Allison D" wrote:
> 
> Hello to all in histoland.  I need help in locating a cleaning acid
solution
> for cleaning glassware.  We are having a problem with our GMS stain.  I
> think it has something to do with the glassware.  Thanks in advance
> Allison Scott
-------------------------



------------------------------

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