Hi Nick,
Whenever we use power tools to work on bone in theatre, we use cold
syringing with saline;
a) to prevent thermal necrosis of bone
b) to prevent the 'spray' dust that you mention

Hope this helps

-----Original Message-----
From: HistoNet Server [mailto:histonet@pathology.swmed.edu]
Sent: Friday, October 25, 2002 10:00 PM
To: HistoNet Server
Subject: Daily Digest





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

Date: 25 Oct 2002 00:45:05 -0500
From: antje.marcantonio@pharma.novartis.com
Subject: Re: CD3 rat


Tim

we also use the clone 3-12 for mice and monkeys, for rat FFPE we have
success with the polyclonal anti-human CD3 from DakoCytomation.
We usually do antigen retrieval with citrate buffer pH 6.0 for 20 min in
microwave. Dilution for the primary is 1:200 , incubation for 60 min.
Hope this helps.

Antje Marcantonio
Novartis Pharma AG
Basle, Switzerland



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

Date: 25 Oct 2002 02:45:50 -0500
From: louise renton 
Subject: Re: Bone sampling

Nick,
Depends on the size of the sample needed and of course the specimen. What
about using a Jamshidi(?sp) needle used for bone marrow sampling? The ones
I've seen make a small core biopsy about 2mm diameter. but possibly there
are larger ones out there. We used to get cores for  metabolic bone studies
that were larger about 5mm, but I have no idea what they used to get them.
How about finding a friendly orthopod who will let you look at his array of
instruments?

Best regards

Louise Renton
Bone Research Unit
MRC
Johannesburg
South Africa
Tel & fax +27 11 717 2298
"Time flies like an arrow, fruit flies like a banana"





>From: Nick Bullough 
>To: "Histonet (E-mail)" 
>Subject: Bone sampling
>Date: Thu, 24 Oct 2002 15:13:04 +0100
>
>Can anyone help with a nice little problem I have?
>
>I need to sample both cortical and cancellous bone for histology and RNA
>extraction. I've seen a dowel cutter that can be attached to a drill to
>take
>cores of bone that will be ideal for what is needed.
>
>My problem is that the health & safety people are very nervous about
>aerosols and other contamination (bone dust). My idea was to enclose the
>cutting in a box or behind a shield as it will be almost impossible to do
>this within a safety cabinet (and there is still the problem of spray from
>the cutter.
>
>How do other people deal with bone sampling? any good ideas very gratefully
>received!
>
>Best regards
>
>Nick
>
>Nick Bullough BSc(Hons) AIBMS
>Scientist
>Bioresources
>Pharmagene plc
>2 Orchard Road
>Royston
>Herts SG8 5HD
>UK
>
>nick.bullough@pharmagene.com
>
>
>
>***************************************************************************
***
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>do need to check this e-mail and any attachments to it for viruses as
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Broadband?#160#Dial-up? Get reliable MSN Internet Access.
http://resourcecenter.msn.com/access/plans/default.asp



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

Date: 25 Oct 2002 04:00:54 -0500
From: Phil Bergin 
Subject: Alternate thick-thin cryostat sections

Hi,



We have a Leica CM1800 cryostat which is great and serviced regularly
with no problems.  All of our biopsies are embedded in OCT and we
generally section at about 8 microns in thickness.  We often get
sections which come out with every other section being thin, followed by
one which is apparently the correct thickness, followed by another thin
section and so on.  This doesnit happen on every biopsy, but on enough
for it to be a regular occurrence.  Is this normal?  Or is it something
to do with the way the specimens are being prepared?  According to our
service rep it is something that just happens and should be ignored.



Thanks for any advice,



Phil



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



Philip Bergin

Goteborg University

Department of Medical Microbiology and Immunology

Box 435, SE-405 30 Goteborg, Sweden

Phone: +46-31-3424727

Fax: +46-31-826976






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

Date: 25 Oct 2002 04:45:57 -0500
From: Lee & Peggy Wenk 
Subject: Re: H pylori and special stains

We do three levels on all gastric biopsies, with one extra unstained slide
at each level.

We do Diff Quik on the gastric biopsies that come down with the a pre-biopsy
"diagnosis" of ulcer (equivalent of "rule out ulcer/H. pylorii"). An
additional extra slide at the 2nd level is cut for this stain. (this still
leaves one unstained at the 2nd level, just in case. . . )

If the r/o is for cancer or any other diagnosis besides the ulcer, we still
cut 3 levels H&E with one extra unstained at each level, but do not do the
Diff Quik.

If the pathologist, upon seeing the H&E of a case that did not have the "r/o
ulcer", sees something that suggests ulcer, they can come back and ask us to
do the Diff Quik on one of the extra unstained slides from the levels.

If the Diff Quik does not demonstrate H. pylori, and the pathologist is
still convinced that the patient must have it, we use one of the extra
unstained slides to do a Steiner and Steiner.

Once is a great while, the St & St does not demonstrate H. pylorii, and it
the pathologist still suspects ulcer, so the pathologist requests IHC to be
done on another additional unstained slide.

Yes, H. pylorii does seem to skip around, and is not necessarily seen at all
levels.

Hope this helps.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073


- ----- Original Message -----
From: 
To: ; 
Sent: Thursday, October 24, 2002 1:27 PM
Subject: H pylori and special stains


> Hi all,
>     Does your lab automatically perform special stains for H pylori on all
> gastric biopsy material as a matter of routine or do you wait and perform
> them at the discretion of the pathologist after the H&E-stained sections
have
> been seen?  Has anyone had experience with seeking an annual 'standing
order'
> from gastroenterologist groups desiring these special stains on all
gastric
> biopsies?
> Thanks,
> K Whittenburg
>



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

Date: 25 Oct 2002 05:45:44 -0500
From: Augusto Marcuzzi 
Subject: American Optical Spare parts

 Does anyone where I can get spare parts for American Optical tissue
processor (T/P8000) and 820 Microtome
Thanks in advance.
augustomarcuzzi@tutopia.com



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

Date: 25 Oct 2002 06:45:57 -0500
From: Anne Undersander 
Subject: VEGF response


Thanks for responses on supplier for VEGF antibody.  One more questions.
What
type of tissue would you use as a positive control?

Thanks.
Anne Undersander HT(ASCP)
Vet. Med./Animal Science
University of Minnesota



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Thanks for responses on supplier for VEGF
antibody.  One more questions.  What type of tissue would you
use
as a positive control?
 
Thanks. 
Anne Undersander HT(ASCP)
Vet. Med./Animal
Science
University of Minnesota

- --Boundary_(ID_tvUMQQ/mWGm/XPWCXgKU2w)--


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

Date: 25 Oct 2002 07:30:44 -0500
From: Bryan Hewlett 
Subject: Re: Mitochondrial immunostaining (long)

Alessandra,

As usual, John is right on the money!

Appropriate, complete fixation is crucial for the demonstration of
mitochondria.

I would also recommend Baker's acid hematin for straight LM.
However, the Altmann - Metzner stain is absolutely brilliant on paraffin
sections. I still have the slides I stained (Gulp) 45years ago!
There is one added bonus to this technique for your purposes. The Acid
Fuchsine used in Altmann's is stunningly fluorescent by confocal microscopy.
In addition, the photobleaching is negligible.


Bryan R.Hewlett



- ----- Original Message -----
From: "J. A. Kiernan" 
To: "Alessandra Livraghi" 
Cc: "HistoNet Server" 
Sent: Friday, October 25, 2002 12:03 AM
Subject: Re: Mitochondrial immunostaining (long)


> Alessandra Livraghi wrote:
> > Here I am again. Some time ago I was asking for some histochemical
method to
> > label mitochondria in  human tissues...I got some good advices and the
> > staining with SDH and NBT works pretty well.
> > Now I'm back on the same issue, but I'm looking for some GOOD advices
about
> > antibodies to stain mitochondria in paraffin embedded and/or frozen
sections
>
> The very brief (4 min) formaldehyde treatment of your
> cryosections will not fix anything, and the 100% alcohol
> that follows will wreck the organelles. Mitochondria are
> fragile little things, destroyed or deformed by some fixatives
> and destroyed or deformed by water etc if you don't fix them.
>
> Dehydrogenase histochemistry on unfixed or almost unfixed
> sections certainly shows mitochondrial enzyme activity, but
> the sites of deposition of the stain do not necessarily
> correspond to the mitochondria that you'd see in an electron
> micrograph or in a cell vitally stained with janus green B or
> one of the modern vital fluorochromes, or with a classical LM
> stain for properly fixed mitochondria. These statements apply
> equally to immunostained mitochondrial antigens in inadequately
> fixed cells.
>
> The best fixatives for mitochondria in light microscopy are
> archaic mixtures containing potassium dichromate and osmium
> tetroxide applied to minute fragments of tissue, and thoroughly
> washed out before processing into paraffin. Manfred Gabe, who
> was an expert, wrote in his 1976 book that the clearing agent
> should be cedarwood oil (which is viscous and not all removed
> during infiltration with wax). I don't know what all this does
> to the antigens; probably noone has tried to find out.
>
> A neoclassical approach is to fix in neutral formaldehyde (added
> calcium ions improve the picture) or glutaraldehyde (or a mixture
> of the two), and post-chrome the specimen. After some days in an
> aqueous dichromate solution the hydrophilic phospholipids (those
> of mitochondria and myelin) are rendered insoluble in organic
> solvents and therefore stainable in paraffin sections.
>
> I have used Bensley's copper-haematoxylin and Baker's acid
> haematein for this purpose, and I recommend the latter because
> it's easy to do and is a thoroughly investigated and fairly
> well understood histochemical method.
>
> I've never tried Altmann's stain, but have been told that the
> bright red mitochondria on a pale yellow background are
> impressive. Baker's acid haematein gives blue mitochondria
> with a dirty-yellow background. The  yellowy background is
> due to haematein acting as an anionic dye in its own right,
> without any "mordant" metal ions in the staining solution. The
> blue color is that of the complex of haematein with the bound
> chromium in the mitochondrial lipids. It's much the same
> colour as the aluminium-haematein complex in cell nuclei in
> sections stained with H & E (or is it H&E as a single wordlet?).
>
> There's plenty of published work in the field of mitochondrial
> staining. If you have access to a library I'll be happy to
> provide references to books, peer-reviewed original papers and
> review articles in this field.
>
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan/
>
>




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

Date: 25 Oct 2002 07:33:59 -0500
From: Fabien Fuente 
Subject: spurr



Fabien Fuente
Natural Implant
Case Postale 908
Parc Luminy Entreprise
163 Av de Luminy
13288 Marseille Cedex 9
Tel: 04 91 82 65 23
Fax: 04 91 82 65 21



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contained in the following MIME Information.
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Fabien Fuente
Natural Implant
Case
Postale
908
Parc Luminy Entreprise
163 Av de Luminy
13288 Marseille Cedex
9
Tel: 04 91 82 65 23
Fax: 04 91 82 65 21

- --Boundary_(ID_n6ggsHjdbIiImZ+8Y0FSIQ)--


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

Date: 25 Oct 2002 07:34:15 -0500
From: Bryan Hewlett 
Subject: Re: Alternate thick-thin cryostat sections

Phil,

I've had this happen periodically.
I believe this is due to a rapid cutting action when the lubricants are
sticky and the advance mechanism just doesn't keep up!
The solution was to lubricate all sliders with low temp oil, just before
each days use, operate the microtome to distribute the oil, then proceed to
cut sections using a slow action.

Bryan R. Hewlett

- ----- Original Message -----
From: "Phil Bergin" 
To: 
Sent: Friday, October 25, 2002 4:48 AM
Subject: Alternate thick-thin cryostat sections


Hi,



We have a Leica CM1800 cryostat which is great and serviced regularly
with no problems.  All of our biopsies are embedded in OCT and we
generally section at about 8 microns in thickness.  We often get
sections which come out with every other section being thin, followed by
one which is apparently the correct thickness, followed by another thin
section and so on.  This doesn't happen on every biopsy, but on enough
for it to be a regular occurrence.  Is this normal?  Or is it something
to do with the way the specimens are being prepared?  According to our
service rep it is something that just happens and should be ignored.



Thanks for any advice,



Phil



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



Philip Bergin

G#246#teborg University

Department of Medical Microbiology and Immunology

Box 435, SE-405 30 G#246#teborg, Sweden

Phone: +46-31-3424727

Fax: +46-31-826976










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

Date: 25 Oct 2002 08:00:11 -0500
From: Ian Montgomery 
Subject: Fish muscle.


A question for the marine histologists out there.
         Fish muscle, what are the histological characteristics and does it
have any features which distinguish it from other species.
Ian.

Dr. Ian Montgomery,
Histotechnology,
Graham Kerr Building,
Institute of Biomedical & Life Sciences,
University of Glasgow,
Glasgow,
G12 8QQ.
Tel: 0141 339 8855
Office: 4652
Lab: 6644.
e-mail: ian.montgomery@bio.gla.ac.uk



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contained in the following MIME Information.
********************************************


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A question for the marine histologists out there.

        Fish
muscle, what are the histological characteristics and does it have any
features which distinguish it from other species. 

Ian.  


Dr. Ian Montgomery,

Histotechnology,

Graham Kerr Building,

Institute of Biomedical & Life Sciences,

University of Glasgow,

Glasgow,

G12 8QQ.

Tel: 0141 339 8855 

Office: 4652

Lab: 6644.

e-mail: ian.montgomery@bio.gla.ac.uk

- --Boundary_(ID_j6hIE9/oWeao1sGgbdBMAA)--


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

Date: 25 Oct 2002 08:16:10 -0500
From: Tom McNemar 
Subject: RE: H pylori and special stains

Hi,

We automatically do H.Pylori on all gastrics.  We also do Alcian-Blue PAS on
all esophageal biopsies and PAS & Retic on all bone marrows.

As for a standing order..... Are special stains not at the descretion of the
pathologist?

Tom Mc Nemar HT(ASCP)
Histology Supervisor
Licking Memorial Hospital
Newark, Ohio



- -----Original Message-----
From: Instaken@aol.com [mailto:Instaken@aol.com]
Sent: Thursday, October 24, 2002 1:28 PM
To: Pathassistants@yahoogroups.com; histonet@pathology.swmed.edu
Subject: H pylori and special stains


Hi all,
    Does your lab automatically perform special stains for H pylori on all
gastric biopsy material as a matter of routine or do you wait and perform
them at the discretion of the pathologist after the H&E-stained sections
have
been seen?  Has anyone had experience with seeking an annual 'standing
order'
from gastroenterologist groups desiring these special stains on all gastric
biopsies?
Thanks,
K Whittenburg




The Information contained in this e-mail message is privileged and
confidential, and is intended for the use of the addressee and no one else.
If you are not the intended recipient, please do not read or use this e-mail
message and notify the sender of the mistaken transmission.


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

Date: 25 Oct 2002 08:16:45 -0500
From: "Johnson, Teri" 
Subject: RE: Alternate thick-thin cryostat sections

Phil,

This is not normal.  It can sometimes indicate something is loose on the
microtome.  I find that incorrect knife angle will produce thick/thin
sectioning.  Try changing the knife angle at small increments and see if
that
helps.

Teri Johnson
Stowers Institute for Medical Research
Kansas City, MO

- -----Original Message-----
From:   Phil Bergin [mailto:philip.bergin@microbio.gu.se]
Sent:   Fri 10/25/2002 3:48 AM
To:     Histonet@pathology.swmed.edu
Cc:
Subject:        Alternate thick-thin cryostat sections

Hi,



We have a Leica CM1800 cryostat which is great and serviced regularly
with no problems.  All of our biopsies are embedded in OCT and we
generally section at about 8 microns in thickness.  We often get
sections which come out with every other section being thin, followed by
one which is apparently the correct thickness, followed by another thin
section and so on.  This doesn=92t happen on every biopsy, but on enough
for it to be a regular occurrence.  Is this normal?  Or is it something
to do with the way the specimens are being prepared?  According to our
service rep it is something that just happens and should be ignored.



Thanks for any advice,



Phil



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



Philip Bergin

G=F6teborg University

Department of Medical Microbiology and Immunology

Box 435, SE-405 30 G=F6teborg, Sweden

Phone: +46-31-3424727

Fax: +46-31-826976










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

Date: 25 Oct 2002 08:23:15 -0500
From: rkline@emscience.com
Subject: Re: Gram Stain


Sandi,
There is a procedure for a Modified Brown and Brenn in the Theory and
Practice of Histotechnology book.  Do you have it?  The method uses
tartrazine.  Works great.  If you need it, let me know.

Rande Kline
EM Science




                      Snobird75@aol.com

                                               To:
histonet@pathology.swmed.edu
                      10/24/2002 07:55         cc:

                      PM                       Subject:  Gram Stain









Dear Friends:
Does someone have a procedure for doing a gram stain, where you don't use
the
50% solution of xylene and picric acid (Bouins).
I know there is a procedure out there. I just don't remember it.
Thanks for you help
SAndi Miller HT
USAMRICD Research
Maryland







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

Date: 25 Oct 2002 09:00:13 -0500
From: Instaken@aol.com
Subject: Re: H pylori and special stains

Hello Pam, Tom and others,
    Thanks for the input on the prospective vs retrospective ordering of
special stains for H pylori on gastric biopsies.  The info has been most
helpful and I see the practice is not uniform from group to group.
    My group is a little split on a blanket prospective special stain order
vs a bit more discretionary approach and this was the reason for my inquiry.
     Yes, I do realize pathologists can self-refer but of course that could
be seen as abuse depending on how contributory the stain is.  I don't want
to
touch off a string of defensive posts on this matter - I am just exploring a
bit.  I did notice this very topic also debated on Path-L.  Some argue well
for prospective H pylori staining as do those against the practice.  I am
trying to balance thinking on the part of those seeing dollar signs, those a
bit more cost conscious and all shades of gray in between - again making no
accusations of my own for those of you who came down on one side or the
other.
    Thanks again for your help.
K Whittenburg


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

Date: 25 Oct 2002 09:46:00 -0500
From: "Edwards, R.E." 
Subject: CAM 5.2


Wanted  a  supplier  of  CAM5.2....................thanks
                                                           Richard  Edwards
                                                                MRC
TOXICOLOGY
UNIT......U.K.............


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

Date: 25 Oct 2002 11:01:05 -0500
From: Fernando Capela e Silva 
Subject: Ziehl-Nielsen staining

I see in an abstract that for histopathological diagnosis of lead poisoning
the authors suggested Ziehl-Nielsen staining for the presence of acid fast
inclusions in osteoclasts. In books and in the Intenet I only found
Ziehl-Nielsen for microbiology/Bacilli/mycobacteriosis. The Ziehl-Nielsen
staining could be the same ( I haven't the original paper)? Can someone help
me?

With compliments

* * * * * * * * * * * * * * * * * * * * * * * * *
Fernando Capela e Silva
Laborat#243#rio de Biologia da Conserva#231##227#o
Departamento de Biologia
Universidade de ...vora
Apartado 94
7002-554 ...vora
PORTUGAL

Phone: +351-266 760 800
Fax: +351-266 711 231
Email: fcs@uevora.pt

http://evunix.uevora.pt/~fcs/FernandoCapelaSilva.htm




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

Date: 25 Oct 2002 11:01:19 -0500
From: james.zimmerman@pharma.novartis.com
Subject:

Has anyone had any problems with lifting off cells for LCM that have been
picked up on SuperFrost Plus Slides?  How

did you resolve the problem?

Also does anyone have any suggestions for keeping fresh frozen cyrostat
sections on any kind of slide.



                         Thanks,

                             JPZ



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

Date: 25 Oct 2002 11:01:35 -0500
From: "Dawson, Glen" 
Subject: RE: CAM 5.2

Richard,

Becton Dickinson

Cat. #349205.

Proteinase K retrieval 4 min.
LSAB2 Detection (DAKO)
Dilution:  1:50 for 30 min.

Works very well.


Good Luck,

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



- -----Original Message-----
From: Edwards, R.E. [mailto:ree3@leicester.ac.uk]
Sent: Friday, October 25, 2002 9:33 AM
To: histonet@pathology.swmed.edu
Subject: CAM 5.2



Wanted  a  supplier  of  CAM5.2....................thanks
                                                           Richard  Edwards
                                                                MRC
TOXICOLOGY UNIT......U.K.............


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

Date: 25 Oct 2002 11:30:33 -0500
From: "Frederick C. Monson" 
Subject: Re: calcium formalin and coacervates

It is with temerity that I launch into an amplification of a Kiernan message
with something chemical (I have done that without pause in the past), but I
think that this brief addition (NOT a correction!) might be of interest to
all that are interested in this thread.

A look at a modern application that I found while looking for an external
and relevant reference to coacervation, which I recall (sans reference) is
what the Ca+2 does, in Lillie's formulation, after it drops its 2 -OAc's off
to tie up protons.

URL:  http://www.swri.edu/3pubs/ttoday/summer95/microeng.htm

Regards and soap to all,

Fred Monson
Quanta400-D7610-WCUPA
SS031, Schmucker II Science Center
West Chester University
West Chester, PA, 19383
Dr. Frederick C. Monson
Phone:  610-738-0437
eMail:  fmonson@wcupa.edu

- ----- Original Message -----
From: "J. A. Kiernan" 
To: "Miller, Yvette" 
Cc: 
Sent: Thursday, October 24, 2002 4:31 PM
Subject: Re: calcium formalin


> Formal-calcium is intended to increase the preservation
> of phospholipids, for subsequent demonstration in
> frozen sections or, following chromation, in paraffin
> sections. Lillie's formulation with calcium acetate is
> perfectly OK as a regular fixative for paraffin work.
> Wash it out with water, then you won't get insoluble calcium
> salts precipitating out in the solvents. (This should be
> less of a problem than with phosphate buffered formaldehyde
> because calcium acetate is soluble in alcohol, unlike the
> Na and K phosphates.)
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan/
>
>
> "Miller, Yvette" wrote:
> >
> >
> > Topic: calcium formalin
> > Has anyone ever used this as a fixative for fatty tissue? Will it clog
> your
> > processor ? Can it be used for processing tissue for paraffin
> embedding ?
> >
> > Local Animal Hospital,
> > Yvette, MA
>
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan/



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

Date: 25 Oct 2002 12:00:19 -0500
From: Ryan Blair Rountree 
Subject: Re: Alternate thick-thin cryostat sections

I have also had this problem with my machine.  Two reasons we've come up
with are 1) something is loose somewhere.  If the knife, knife holder,
block, or microtome head isn't nice and snug, this will definately do it.
Our microtome head as several alan-wrench-screws that can be tightened so
that when you lock the head in the correct orientation it is held very
tightly in place.  Every few months, the head on our machine gets loose,
even to the point where you can readily move it by hand in the lock
position. If you unscrew the long screw that holds the block in
place, the face of the microtome head comes off and you can easily tighten
2 screws for this.  There are others as well though, and you should really
have this done by your service rep.  Just make sure that everything you
can tighten starts tight and stays tight throughout the run!
2) There can be scratches on the "telescoping arm" that extends the
microtome head with each section.  If these are bad enough, the head can
catch on them occasionally as it advances.  This however is a major
repair.  It might cost $2-3000!  In our case there were scratches here and
since we had a service contract it was no big deal to repair.  But the
price of the repair was basically the price of the service contract!
Anyway, I haven't been having this problem any more, but I think the main
reason is that everything is very tight and stable now.  Start with that!

 On Fri, 25 Oct 2002, Phil
Bergin wrote:

> Hi,
>
>
>
> We have a Leica CM1800 cryostat which is great and serviced regularly
> with no problems.  All of our biopsies are embedded in OCT and we
> generally section at about 8 microns in thickness.  We often get
> sections which come out with every other section being thin, followed by
> one which is apparently the correct thickness, followed by another thin
> section and so on.  This doesnit happen on every biopsy, but on enough
> for it to be a regular occurrence.  Is this normal?  Or is it something
> to do with the way the specimens are being prepared?  According to our
> service rep it is something that just happens and should be ignored.
>
>
>
> Thanks for any advice,
>
>
>
> Phil
>
>
>
> -----------------------------------------------------------------
>
>
>
> Philip Bergin
>
> Goteborg University
>
> Department of Medical Microbiology and Immunology
>
> Box 435, SE-405 30 Goteborg, Sweden
>
> Phone: +46-31-3424727
>
> Fax: +46-31-826976
>
>
>
>
>

Ryan Rountree
Department of Developmental Biology
B301, Beckman center
279 Campus Drive
Stanford, CA 94305
(650) 725-7599
Fax: (650) 725-7739



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

Date: 25 Oct 2002 12:30:21 -0500
From: Ryan Blair Rountree 
Subject: LacZ activity in decalcified bone

Hello again, and thanks to everyone that has answered other questions from
me!

Does anyone have a protocol for decalcifying tissue that maintains LacZ
activity?  Especially in mouse limbs?  I get really strong lacZ activity
cryosectioning calcified young mouse limbs (up to 2 weeks old) by fixing
in 4% paraformaldehyde for 4 hours, followed with 15% sucrose for an hour,
then 30% sucrose overnight and OCT freezing.  This time I simply added a
step after fixation of 10% EDTA pH7.4 for 3 days, followed by the sucrose
and OCT freezing (ALL steps done at 4 degrees C).  But the lacZ activity
was totally dead.  Any suggestions?  THANK YOU!!

Ryan

Ryan Rountree
Department of Developmental Biology
B301, Beckman center
279 Campus Drive
Stanford, CA 94305
(650) 725-7599
Fax: (650) 725-7739




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

Date: 25 Oct 2002 13:15:14 -0500
From: Abizar Lakdawalla 
Subject: RE: Mitochondrial immunostaining


bcl-2 is a protein that is located in the mitochondria and I know that there
are quite a few good antibodies out there (including the ones we provide).
In most paraffin sections the staining will appear as "cytoplasmic" with
colorimetric end-points (BCIP/NBT gives the highest resolution as compared
to AEC or DAB). To see at higher resolution you will need to stick to
fluorescent end-points.
Hope this does not muddy the waters even more,
Abizar

www.biogenex.com
www.innogenex.com


>
> -----Original Message-----
> From: Alessandra Livraghi
> To: HistoNet Server
> Sent: 10/24/2002 5:59 PM
> Subject: Mitochondrial immunostaining
>
> Hallo everybody!
> Here I am again. Some time ago I was asking for some histochemical
> method to
> label mitochondria in  human tissues...I got some good advices and the
> staining with SDH and NBT works pretty well.
> Now I'm back on the same issue, but I'm looking for some GOOD advices
> about
> antibodies to stain mitochondria in paraffin embedded and/or frozen
> sections
> of human lung.
> I would like to reproduce the specific stain I see using the
> histochemical
> method with an immunofluorescence technique (I'll do confocal
> microscopy
> on
> these samples)
> For immunofluorescence staining, I've tryed three different antibodies
> (mHSP70 from Affinity Bioreagents, Cyt b from SantaCruz and
> anti-OxPhos
> Complex V inhibitor protein from Molecular Probes), with poor results.
> Now
> I'm wondering if I'm doing something really wrong (fixation,
> incubations
> time..)...or if it's just bad luck with antibodies.
> I've just came back from the last unsuccessful staining. So I
> decided to
> ask
> for some help from people with more experience than me!
> These are the protocols I'm using:
>
> FROZEN SECTIONS:
> Fixation and permeabilization: 4%PFA, 4 min RT. Wash in PBS.
> 100%EtOH, 2
> min -20C. Wash in PBS.
> Blocking: 20% serum (matched with the species where the secondary
> antibody
> is raisen), 3 hrs RT
> Washing: 3x5min in PBS
> Primary antibody: dil 1:100 or 1:50 in 2%serum/PBS, overnight 4C
> Usually I include a control incubated without primary antibody
> (2%serum/PBS
> alone) and another one incubated with the same concentration of IgG
> (ChromPure from JacksonLab) as my antibody (isotypic control).
> Washing: 3x5min in PBS
> Secondary antibody: Alexafluor 568 dil 1:1000 in 2%serum/PBS,
> 30 min RT,
> in
> the dark
> Washing: 3x5min in PBS
> Mounting with Vectashield Mounting medium (with DAPI)
> Results: my isotypic control is stained exactly as the
> antibody treated
> sample(maybe a little bit less, but nothing significant) !
>
> PARAFFIN EMBEDDED SECTIONS (fixed in 10% NBF overnight)
> Dewaxing through xilene (2x5 min) and EtOH 100% (2x3 min),
> 95%(2x3 min),
> 70%(1x3 min)
> Rehydration: PBS, 30 min
> Permeabilization: 0.1%Triton in PBS, 10 min RT. Wash in PBS.
> Blocking: 20% serum (matched with the species where the secondary
> antibody
> is raisen), 3 hrs RT
> From now on, everything is the same as in the protocol for frozen
> sections...including the results! Actually, in this case I've
> tried also
> to
> retrieve antigens by boiling the sections in 10mM Na citrate pH 6.00,
> but
> nothing changed in the final results.
>
> This is the status of my "art". I'm really disappointed!
> If somebody  has a direct experience in immunostaining of
> mitochondria,
> I'll
> greatly appreciate any help!
> Sorry for have been taking so much time of yours,
> Alessandra
>
>
> Alessandra Livraghi, PhD
> Cystic Fibrosis Center- UNC Chapel Hill
> 7013 Thurston Bowles Bldg.
> CB#7248
> Chapel Hill NC 27599
> tel: 919-966-7045
> fax: 919-966-7524
>
>



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bcl-2 is a protein that is located in the mitochondria and I
know that there are quite a few good antibodies out there (including the
ones
we provide). In most paraffin sections the staining will appear as
"cytoplasmic" with colorimetric end-points (BCIP/NBT gives the
highest resolution as compared to AEC or DAB). To see at higher resolution
you
will need to stick to fluorescent end-points.

Hope this does not muddy the waters even more,

Abizar


www.biogenex.com

www.innogenex.com




> 

> -----Original Message-----

> From: Alessandra Livraghi

> To: HistoNet Server

> Sent: 10/24/2002 5:59 PM

> Subject: Mitochondrial immunostaining

> 

> Hallo everybody!

> Here I am again. Some time ago I was asking for some
histochemical

> method to

> label mitochondria in  human tissues...I got some
good advices and the

> staining with SDH and NBT works pretty well.

> Now I'm back on the same issue, but I'm looking for
some
GOOD advices

> about

> antibodies to stain mitochondria in paraffin embedded
and/or frozen

> sections

> of human lung.

> I would like to reproduce the specific stain I see
using
the

> histochemical

> method with an immunofluorescence technique (I'll do
confocal 

> microscopy

> on

> these samples)

> For immunofluorescence staining, I've tryed three
different antibodies

> (mHSP70 from Affinity Bioreagents, Cyt b from
SantaCruz
and 

> anti-OxPhos

> Complex V inhibitor protein from Molecular Probes),
with
poor results.

> Now

> I'm wondering if I'm doing something really wrong
(fixation, 

> incubations

> time..)...or if it's just bad luck with
antibodies.

> I've just came back from the last unsuccessful
staining.
So I 

> decided to

> ask

> for some help from people with more experience than
me!

> These are the protocols I'm using:

> 

> FROZEN SECTIONS:

> Fixation and permeabilization: 4%PFA, 4 min RT. Wash
in
PBS. 

> 100%EtOH, 2

> min -20C. Wash in PBS.

> Blocking: 20% serum (matched with the species where
the
secondary

> antibody

> is raisen), 3 hrs RT

> Washing: 3x5min in PBS

> Primary antibody: dil 1:100 or 1:50 in 2%serum/PBS,
overnight 4C

> Usually I include a control incubated without primary
antibody

> (2%serum/PBS

> alone) and another one incubated with the same
concentration of IgG

> (ChromPure from JacksonLab) as my antibody (isotypic
control).

> Washing: 3x5min in PBS

> Secondary antibody: Alexafluor 568 dil 1:1000 in
2%serum/PBS, 

> 30 min RT,

> in

> the dark

> Washing: 3x5min in PBS

> Mounting with Vectashield Mounting medium (with
DAPI)

> Results: my isotypic control is stained exactly as the


> antibody treated

> sample(maybe a little bit less, but nothing
significant)
!

> 

> PARAFFIN EMBEDDED SECTIONS (fixed in 10% NBF
overnight)

> Dewaxing through xilene (2x5 min) and EtOH 100% (2x3
min), 

> 95%(2x3 min),

> 70%(1x3 min)

> Rehydration: PBS, 30 min

> Permeabilization: 0.1%Triton in PBS, 10 min RT. Wash
in
PBS.

> Blocking: 20% serum (matched with the species where
the
secondary

> antibody

> is raisen), 3 hrs RT

> From now on, everything is the same as in the protocol
for frozen

> sections...including the results! Actually, in this
case
I've 

> tried also

> to

> retrieve antigens by boiling the sections in 10mM Na
citrate pH 6.00,

> but

> nothing changed in the final results.

> 

> This is the status of my "art". I'm really
disappointed!

> If somebody  has a direct experience in
immunostaining of 

> mitochondria,

> I'll

> greatly appreciate any help!

> Sorry for have been taking so much time of
yours,

> Alessandra

> 

> 

> Alessandra Livraghi, PhD

> Cystic Fibrosis Center- UNC Chapel Hill

> 7013 Thurston Bowles Bldg.

> CB#7248

> Chapel Hill NC 27599

> tel: 919-966-7045

> fax: 919-966-7524

> 

> 





- --Boundary_(ID_uRII/o5W7LYuGx8P6wiDqw)--


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

Date: 25 Oct 2002 13:15:31 -0500
From: "Wright, Clarissa B  CIV" 
Subject: RE: CAM 5.2

Richard,
        Try Becton-Dickinson
                Anti-cytokeratin (Cam 5.2)
                Cat#349205


Clarissa B. Wright, HT
Laboratory Dept EDA 13
Naval Medical Center San Diego
34800 Bob Wilson Drive
San Diego Ca 92134-1305


- -----Original Message-----
From: Edwards, R.E. [mailto:ree3@leicester.ac.uk]
Sent: Friday, October 25, 2002 7:33 AM
To: histonet@pathology.swmed.edu
Subject: CAM 5.2



Wanted  a  supplier  of  CAM5.2....................thanks
                                                           Richard  Edwards
                                                                MRC
TOXICOLOGY UNIT......U.K.............


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

Date: 25 Oct 2002 14:15:13 -0500
From: Jo Dee Fish 
Subject: Need processing help!!

Dear Histonetters,
We have a bit of a problem... our paraffin processor did not run
overnight, and the tissues sat dry all night.  We currently are
storing them in 70% EtOH until they can be processed.
We would like to have another lab process a load of samples for us.
Unfortunately, I don't know anyone in the area.  I was hoping that
someone in our immediate area, South San Francisco, could call me if
they can help out, or know of a lab that will.  My phone number is:
415-695-3720.
Thanks so much,
Desperate in SF,
Jo Dee Fish

#003#Jo Dee Fish
Research Technologist II
Gladstone Institue of Cardiovascular Disease

Telephone: (415) 695-3720
Fax: (415) 285-5632
E-mail: jfish@gladstone.ucsf.edu

Mailing address:
Gladstone Instutes
P.O. Box 419100
San Francisco, CA 94141-9100


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

Date: 25 Oct 2002 14:30:52 -0500
From: Jennifer Hofecker 
Subject: call for abstracts - Tennessee meeting

 Hi everyone,

The Tennessee Society of Histotechnology is now
accepting workshop abstracts for our spring meeting to
be held March 6-8, 2003, in Gatlinburg which is
located in the scenic Smokey Mountains.

If you think you might be interested in presenting a
workshop or have questions, please contact me at the
number below or by email.  I would especially like to
hear from any fellow Tennesseans that might be willing
to present.

Thank you so much.
Hope to see you in Gatlinburg!

Jennifer Hofecker

OUR Lab
Nashville, TN
ph. (615)874-0410
fax (615)232-8009

__________________________________________________
Do you Yahoo!?
Y! Web Hosting - Let the expert host your web site
http://webhosting.yahoo.com/


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

Date: 25 Oct 2002 15:15:18 -0500
From: Snobird75@aol.com
Subject: Re: Gram Stain

Dear John
After you use crystal violet, Gram Iodine  and counterstain, towards the end
of the stain you have to use a 50% solution of xylene and acetone. Then take
it into the xylene and coverslip. There is a step where Bouins and Formalin
mixture is used.
I am trying to get rid of the xylene/acetone step. I don't have the
procedure
at home with me. Its just a stain that the lab uses where I work.
Thank you
sandi miller HT
USAMRICD Research
Maryland


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

Date: 25 Oct 2002 17:30:25 -0500
From: Jeff Silverman 
Subject: Factor XIIIa

Charles,
Calbiochem has it, rabbit anti-human factor XIIIa that is,  the price has
increased to 160 dollars or so for 1 ml. I remember buying my first ml for
35 dollars in 1997 or so. Wrote a lot of papers using that first ml. I used
the Calbiochem product at 1:400 in PBS with 1% BSA after 20-30 minute
trypsin (Sigma tablets) digestion  at 37 deg C for FFPE tissues. Biogenex
sells a prediluted and concentrate polyclonal as well (concentrate is best
at 1:30 to 1:60 in PBS, no carrier protein necessary they add it already-
they recommend citrate HIER). Biocare Medical also lists FXIIIa and
Novacastra has one too.
Hope this helps.
Jeff Silverman HT HTL QIHC
Southside Hospital
Bay Shore NY USA



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

Date: 25 Oct 2002 23:15:15 -0500
From: Paul Bradbury 
Subject: Re: Gram Stain

Hi. I think the method you are talking about is the Brown-Hopp's variant of
the Gram stain. We use this method regularly to demonstrate H. pylori in
gastric biopsies.

After doing a more-or-less conventional Gram stain, the section is treated
with:
- - acetone (to dehydrate it)
- - acetone-picric acid (to stain the background yellow)
- - acetone/xylene (to "wash off" excess picric acid, and begin clearing)
- - xylene (to complete clearing prior to mounting)

Why do you want to get rid of the acetone-xylene step?

If you take the section from acetone-picric acid straight into pure xylene,
any residual picric acid may precipitate out (picric acid is essentially
insoluble in xylene).

It will probably be quite okay to take sections from acetone-picric acid
into a "wash" with pure acetone (just to remove any excess picric acid).
This will also complete the dehydration. Then treat with a couple of changes
of xylene and mount.


Paul Bradbury, FIMLS, ART
Kamloops, BC Canada





- ----Original Message Follows----
From: Snobird75@aol.com
To: jkiernan@uwo.ca, histonet@pathology.swmed.edu
Subject: Re: Gram Stain
Date: Fri, 25 Oct 2002 15:53:43 -0400 (EDT)

Dear John
After you use crystal violet, Gram Iodine  and counterstain, towards the end
of the stain you have to use a 50% solution of xylene and acetone. Then take
it into the xylene and coverslip. There is a step where Bouins and Formalin
mixture is used.
I am trying to get rid of the xylene/acetone step. I don't have the
procedure
at home with me. Its just a stain that the lab uses where I work.
Thank you
sandi miller HT
USAMRICD Research
Maryland


_________________________________________________________________
Surf the Web without missing calls! Get MSN Broadband.
http://resourcecenter.msn.com/access/plans/freeactivation.asp



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

Date: 25 Oct 2002 23:15:33 -0500
From: "Ms. Evelyn Kaplan" 
Subject: Re: H pylori and special stains

We routinely do a Tol Blue/Alcian Yellow in our lab. We reckon it is the
best if you are using the Sydney System to report the gastric biopsies.

Evelyn Kaplan
Dept of Pathology,
Sultan Qaboos University,
Muscat, Oman



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