I am looking for a recipe for acid cleaning glassware to do silver stains,
can anyone help me?

-----Original Message-----
From: histonet-request@lists.utsouthwestern.edu
[mailto:histonet-request@lists.utsouthwestern.edu]
Sent: Tuesday, September 16, 2003 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet digest, Vol 1 #49 - 19 msgs


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Today's Topics:

   1. Re: Bad Plants/Yarrow (sara goldston)
   2. background problem (Jacqueline Miller)
   3. Re: Bouin's fixation/removal of picric acid (Sarah Jones)
   4. eye morphology advice (Margaret Gondo)
   5. ASCP BOR results (Lee & Peggy Wenk)
   6. wage and salary survey (Lee & Peggy Wenk)
   7. Colloidal Iron (lena spencer)
   8. Re: Bouin's fixation/removal of picric acid (John Kiernan)
   9. Re: Colloidal Iron (John Kiernan)
  10. RE: eye morphology advice (Kemlo Rogerson)
  11. Re:  Methacarn/Carnoy's Storage (Barbara Osborn)
  12. IHC on Blood Smears (Jackie.O'Connor@abbott.com)
  13. Re: eye morphology advice (Fred Underwood)
  14. Re: eye morphology advice (Fred Underwood)
  15. Re: IHC on Blood Smears (Fred Underwood)
  16. immunohistochemistry and frozen sections (Vincenzo La Manna)
  17. Re: immunohistochemistry and frozen sections (Phillip Huff)
  18. Talented Histotechs wanted (Tommy Vedilago)
  19. Re: immunohistochemistry and frozen sections
(nick.kirk3@btopenworld.com)

--__--__--

Message: 1
Date: Mon, 15 Sep 2003 12:43:52 -0700 (PDT)
From: sara goldston 
To: "Johnston, Kathy" ,
  "Histonet \(E-mail\)" 
Subject: Re: [Histonet] Bad Plants/Yarrow

--0-349928651-1063655032=:6512
Content-Type: text/plain; charset=us-ascii

I use to be a naturalist in high school and yarrrow is suppose to have
medicinal purposes (hopefully you and I are talking about the same plant).
If you crush either the leaves or blooms, cannot recall which, under you
nose you will smell something similar to Vick's vapor rub.  Yarrow has the
same effect as Vick's by opening up the nasal passages.  But more to your
question at hand.  Yarrow is a wild bush so I would assume like any wild and
un-domesticated plant, it will grow quite freely if given the opportunity
to.  If you are from anywhere east of the Mississippi, there has been an
unusual amount of rain not to mention cooler than normal temperatures.  If
your plant was kept outside, it would have loved the weather this season and
flourished!  You may have to break out the pruining shears.  Sorry, I don't
have any other helpful advice.  Try going to a nature reserve, greenhouse or
horticulturalist, and perhaps someone along those lines will have some more
concrete advise.
Sara 

"Johnston, Kathy"  wrote:
While we are talking "Bad Plants"  I would like everyone's opinion on
Yarrow.  I bought 2 plants this spring and they have grown into to very
large although quite stunning plants.  I can't remember it's exact name
right now, but each stem is a different pastel color.  My only concern with
is is that is started out in a 2" pot, and right now I would need a 5 gallon
pail to put it in if I dug it out!
 
I am understanding that is seems quite prolific.  But is it easy to control.
 
Your honest opinions please!!!!
 
Thanks!
 
Kathy




---------------------------------
Do you Yahoo!?
Yahoo! SiteBuilder - Free, easy-to-use web site design software
--0-349928651-1063655032=:6512
Content-Type: text/html; charset=us-ascii

I use to be a naturalist in high school and yarrrow is suppose to have
medicinal purposes (hopefully you and I are talking about the same
plant).  If you crush either the leaves or blooms, cannot recall which,
under you nose you will smell something similar to Vick's vapor rub. 
Yarrow has the same effect as Vick's by opening up the nasal passages. 
But more to your question at hand.  Yarrow is a wild bush so I would
assume like any wild and un-domesticated plant, it will grow quite freely if
given the opportunity to.  If you are from anywhere east of the
Mississippi, there has been an unusual amount of rain not to mention cooler
than normal temperatures.  If your plant was kept outside, it would
have loved the weather this season and flourished!  You may have to
break out the pruining shears.  Sorry, I don't have any other helpful
advice.  Try going to a nature reserve,
greenhouse or horticulturalist, and perhaps so
 meone
 along those lines will have some more concrete advise.
Sara 

"Johnston, Kathy"
<Kathy.Johnston@CLS.ab.ca> wrote:


While we are talking "Bad
Plants"  I would like everyone's opinion on Yarrow.  I bought 2
plants this spring and they have grown into to very large although quite
stunning plants.  I can't remember it's exact name right now, but each
stem is a different pastel color.  My only concern with is is that is
started out in a 2" pot, and right now I would need a 5 gallon pail to put
it in if I dug it out!
 
I am understanding that is
seems quite prolific.  But is it easy to control.
 
Your honest opinions
please!!!!
 
Thanks!
 
Kathy

Do you Yahoo!?

Yahoo!
SiteBuilder - Free, easy-to-use web site design software
--0-349928651-1063655032=:6512--


--__--__--

Message: 2
Date: Mon, 15 Sep 2003 15:02:24 -0500
From: "Jacqueline Miller" 
To: 
Subject: [Histonet] background problem

Hi,

We're having trouble with high background when using Rat IgG1 antibodies =
(4 =B5g/ml) on paraffin-embedded sections of human tissues, with or =
without retrieval procedures.  Does anyone have any advice?

Thank you,

Jacqueline Miller
Research Asst. I
OB/GYN Dept.
University of Texas Southwestern Medical Center
Dallas, TX





--__--__--

Message: 3
Date: Mon, 15 Sep 2003 15:57:36 -0500
From: "Sarah Jones" 
To: 
Cc: ,
Subject: Re: [Histonet] Bouin's fixation/removal of picric acid

I use it on wet tissue, it does not appear that the lithium needs to be
removed.  Maybe I'm not understanding your question.   Sarah
Ref. Theory and Practice of Histotechnology, Sheehan, Hrapchak, page
47.    


>>> John Kiernan  09/14/03 11:54PM >>>
Sarah, do you use the saturated lithium 
carbonate in 70% ethanol on blocks of
tissue or on hydrated paraffin sections
that are yellow?  

If it's for blocks, how do you remove
lithium carbonate from the decolorized
specimens?  
              John Kiernan
-- 
-------------------------
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/ 
___________
Sarah Jones wrote:
> 
> I've always used saturated lithium carbonate in 70% ethanol to
remove
> the picric acid.  I believe it would be faster and more complete
than
> 70% alone.
> 
> Sarah Jones HT(ASCP)
> Dept. of Vet. Anatomy & Public Health
> Histology Lab
> Texas A&M University
> College Station, TX 77843-4458
> phone: 979-845-3177
> fax:  979-458-3499
> 
> >>> "peptolab"  09/13/03 08:32AM >>>
> A 3-5 mm testicular biopsy should be fixed in one to two hours. Be
sure
> to
> rinse out as much yellow picric acid as possible (you can use
running
> water
> or 70% alcohol ). The basophilia of chromatin in the stored block
will
> deteriorate after some time if there is picric acid  residue.
> 
> Jeff Silverman HT HTL QIHC (ASCP)
> Southside Hospital
> Bay Shore NY
>


--__--__--

Message: 4
Date: Mon, 15 Sep 2003 16:28:30 -0500
From: "Margaret Gondo" 
Reply-To: 
To: 
Subject: [Histonet] eye morphology advice

Hi All -

I have a grad student who is interested in preserving the 
morphology of a monkey eyeball once it is removed.  Does anyone 
have any suggestions?  Everything we have tried always results in 
the posterior part of the eyeball caving in.  



Thanks in advance,
Margaret


 



--__--__--

Message: 5
From: "Lee & Peggy Wenk" 
To: "Histonet" 
Cc: 
Date: Mon, 15 Sep 2003 19:10:59 -0400
Subject: [Histonet] ASCP BOR results

For those interested in the pass rates of the HT and HTL and SLS ASCP exams
Jan. - June 2003, please read on. (For those not interested, please press
the DELETE key now.)

Scaled score of 400 is required to pass the exam. For HT and HTL, both the
written (MCQ = Multiple Choice Question) and the practical portions must be
passed.

NAACLS = National Accrediting Agency for Clinical Laboratory Sciences, which
is the accrediting agency for lab training programs.

Total Number non-NAACLS candidates = Total Number taking exam - Total Number
NAACLS students

HISTOTECHNICIAN

HT MCQ
Mean = 428
Range of Scores = 100-761
Total Number Taking Exam = 206
Total Pass = 111 (54%)
Total Number NAACLS students = 24
Total Pass NAACLS students = 18 (75%)
Total Number non-NAACLS candidates = 182
Total Pass non-NAACLS candidates = 93 (51%)

HT Practical
Mean = 446
Range of Scores = 201-889
Total Number Taking Exam = 217
Total Pass = 144 (66%)
Total Number NAACLS students = 24
Total Pass NAACLS students = 20 (83%)
Total Number non-NAACLS candidates = 193
Total Pass non-NAACLS candidates = 20 (64%)

HT COMBINED
Total Number Taking Exam = 349
Total Pass = 141 (40%)
Total Number NAACLS students = 23
Total Pass NAACLS students = 15 (65%)
Total Number non-NAACLS candidates = 326
Total Pass non-NAACLS candidates = 126 (39%)

HT exam first given in 1948. Certified to date = 18,556


HISTOTECHNOLOGIST

HTL MCQ
Mean = 451
Range of Scores = 100-646
Total Number Taking Exam = 43
Total Pass = 32 (74%)
Total Number NAACLS students = 0

HTL PRACTICAL
Mean = 487
Range of Scores = 244-700
Total Number Taking Exam = 54
Total Pass = 43 (80%)
Total Number NAACLS students = 0

HTL COMBINED
Total Number Taking Exam = 78
Total Pass = 39 (50%)
Total Number NAACLS students = 0

HTL exam first given in 1980. Certified to date = 2,070


SPECIALIST IN LABORATORY SAFETY
Mean = 422
Range of Scores = 363-511
Total Number Taking Exam = 7
Total Pass = 3 (43%)

SLS exam first given in 2000. Certified to date = 138

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



--__--__--

Message: 6
From: "Lee & Peggy Wenk" 
To: "Histonet" 
Cc: 
Date: Mon, 15 Sep 2003 19:19:56 -0400
Subject: [Histonet] wage and salary survey

FYI -

The September 2003 issue of "Laboratory Medicine" from ASCP has Part I of
the latest Wage and Salary Survey. This survey is done every other year.
This article shows national averages of all lab disciplines, and then breaks
it down into regional areas. Please remember, your state, or even your part
of the state, may pay higher or lower than these averages.

Things of interest that I found:

- HT and HTL still have the highest vacancy rates of all laboratory
personnel. This is the same as the survey done 2 years ago.

- HTL and MT now have the same salary ranges, on average nationwide. In
other words, on national average, HTL are now being paid the same rate as
MT.

A few of the charts on the national averages can be found on the ASCP Board
of Registry website:
http://www.ascp.org/bor/center/center_research.asp

Thought some people might like to find their issue, or borrow one from a
colleague (or maybe even join ASCP).

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



--__--__--

Message: 7
From: "lena spencer" 
To: 
Date: Mon, 15 Sep 2003 20:58:58 -0400
Subject: [Histonet] Colloidal Iron

Hi All:
One of my Pathologist's has requested a Hale's Colloidal Iron for renal cell
carcinoma chromophobes.  I am familiar with colloidal iron for
mucosubstances, but he feel's that this is not the same stain.   The article
stated that the diagnosis is made from the colloidal iron stain  or from
electron microscopy .  This Pathologist is requesting that I post on the
histonet for additional information or for someone out in histoland who is
performing this procedure.
Your is insight would be greatly appreciated.
Lena




--__--__--

Message: 8
Date: Tue, 16 Sep 2003 00:24:20 -0400
From: John Kiernan 
Reply-To: jkiernan@uwo.ca
To: Sarah Jones 
CC: histonet@pathology.swmed.edu
Subject: Re: [Histonet] Bouin's fixation/removal of picric acid

I didn't phrase my question very clearly, and
should have explained the reason for asking.
 
Previously I've encountered lithium carbonate
for removing picric from sections. It's the 
traditional thing to do after Bouin fixation
when the tissue is still yellow in the wax 
and after hydration of the sections.
You can easily see when all the yellow has
gone. 

With a block you can't see the middle, and 
I was wondering how long it takes until no
more yellow comes out into the liquid. With
70% and higher alcohols alone it seems to take 
for ever (at least with 5 mm and larger specimens; 
I haven't used tiny biopsies after Bouin fixation). 
It would be nice to have an idea how much more 
quickly the picric acid is extracted when the 
70% alc is saturated with Li2CO3.

I asked about removing the Li2CO3 because it's
almost insoluble in alcohol (Merck Index). That
would be 95-100% alc. I guess you rinse the
specimens in more 70% after extracting the 
picric acid, and that removes the Li2CO3 and
you don't get damage from crystals forming in
the tissue.
-- 
-------------------------
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/
_________________________
Sarah Jones wrote:
> 
> I use it on wet tissue, it does not appear that the lithium needs to be
> removed.  Maybe I'm not understanding your question.   Sarah
> Ref. Theory and Practice of Histotechnology, Sheehan, Hrapchak, page
> 47.
> 
> >>> John Kiernan  09/14/03 11:54PM >>>
> Sarah, do you use the saturated lithium
> carbonate in 70% ethanol on blocks of
> tissue or on hydrated paraffin sections
> that are yellow?
> 
> If it's for blocks, how do you remove
> lithium carbonate from the decolorized
> specimens?
>               John Kiernan
> --
> -------------------------
> 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/
> ___________
> Sarah Jones wrote:
> >
> > I've always used saturated lithium carbonate in 70% ethanol to
> remove
> > the picric acid.  I believe it would be faster and more complete
> than
> > 70% alone.
> >
> > Sarah Jones HT(ASCP)
> > Dept. of Vet. Anatomy & Public Health
> > Histology Lab
> > Texas A&M University
> > College Station, TX 77843-4458
> > phone: 979-845-3177
> > fax:  979-458-3499
> >
> > >>> "peptolab"  09/13/03 08:32AM >>>
> > A 3-5 mm testicular biopsy should be fixed in one to two hours. Be
> sure
> > to
> > rinse out as much yellow picric acid as possible (you can use
> running
> > water
> > or 70% alcohol ). The basophilia of chromatin in the stored block
> will
> > deteriorate after some time if there is picric acid  residue.
> >
> > Jeff Silverman HT HTL QIHC (ASCP)
> > Southside Hospital
> > Bay Shore NY
> >


--__--__--

Message: 9
Date: Tue, 16 Sep 2003 01:22:32 -0400
From: John Kiernan 
Reply-To: jkiernan@uwo.ca
To: lena spencer 
CC: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Colloidal Iron

There's just the one Hale's (1946) method, also called
the dialysed iron technique because if you make
the original reagent yourself you have to dialyse 
the ferric hydroxide sol. Detailed instructions are
given in Pearse's Histochemistry (any edition).
There have been several variants of the principal
reagent. In all the techniques the bound ferric
iron is made visible by a Prussian blue reaction
with potassium ferrocyanide.

In Chapter 10 of the latest (2002) edition of
Bancroft & Gamble's "Theory and Practice ..." by
Barbara Totty, the method attributed to Hale (1946)
uses a more easily made colloidal iron sol that
does not need to be dialysed. A more detailed 
reading of Pearse (3rd ed) shows that this is close 
to the reagent of G.Muller (3 papers in Acta Histochem
vol 2, 1955-56, not seen by me; they'll be in
German). This colloidal iron sol quickly became more 
popular than Hale's reagent not only because it was 
easier to make but also because it was more specific 
for acid mucosubstances. The not-dialysed colloidal
iron solution, unlike Hale's, does not stain nuclei
and there is no "background" cytoplasmic blue colour 
in renal tubules.

Both Pearse and Totty point out that colloidal iron
staining does the same job as alcian blue (pH 2.5)
but provides a darker blue product and also some 
nonspecific staining. It's more sensitive and less
clean.

"Hale's colloidal iron" (subject of the original 
enquiry) is probably not an appropriate name for this
technique, which has been significantly improved
since 1946. Pearse seriously questioned its
histochemical
specificity despite recognizing the improvements made
by Muller and several others. 

Alcian blue is easier to use and can be incorporated 
into many other rational mixed staining procedures
for mucosubstances. I know nothing about renal
carcinoma 
chromophobes. If they can be stained with colloidal
iron
they must be basophilic, not "chromophobic."  There are
very few genuinely chromophobic (unstainable)
cytoplasms.
-- 
-------------------------
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/
__________________________________
lena spencer wrote:
> 
> Hi All:
> One of my Pathologist's has requested a Hale's Colloidal Iron for renal
cell
> carcinoma chromophobes.  I am familiar with colloidal iron for
> mucosubstances, but he feel's that this is not the same stain.   The
article
> stated that the diagnosis is made from the colloidal iron stain  or from
> electron microscopy .  This Pathologist is requesting that I post on the
> histonet for additional information or for someone out in histoland who is
> performing this procedure.
> Your is insight would be greatly appreciated.
> Lena


--__--__--

Message: 10
From: "Kemlo Rogerson" 
To: ,
	
Date: Tue, 16 Sep 2003 07:33:44 +0100
Subject: RE: [Histonet] eye morphology advice

Nitrocellulose and celloidin technique. Page 91 of Histopathologic
Technic and Practical Histochemistry. RD Lillie and Harold M.
Fullmer.1976, 1965 McGrew Inc. 1234567890 KPKP 7832109876

48hr Form fixation
dehydrate with successive baths of 35, 50, 65 & 80% alc. 24 hours each.
Open eye
Return to 80% alc. 24 hours
2x 100% alc 24 hours
6 hour bath in equal vols 100% alc and ether
Infiltrate 5-7 days in 10% nitrocellulose in 100% alc.
Infiltrate 5-7 days in 20% nitrocellulose in 100% alc
Place under bell jar and when surface is solid, examine daily, but lower
portion soft flood with chloroform 16-24 hours.
Pour off chloroform and let dry.
Attach to block with 20% nitrocellulose
Let dry and immerse in chloroform for several hours.
24 hour baths of 3:1 chloroform: cedar wood oil, 1:1 of the same, then
pure cedar wood oil.
Cut sections and store in 80% alc.

Dangerous, smelly, time consuming but extremely good fun; definitely a
non-PC technique that works; done it myself as a pup.

Mr Kemlo Rogerson MSc DMS MIBiol CBiol FIBMS
Tel: 01270 877625
Mob: 07830 196072=20
Mobile E-Mail kemlorogerson@3mail.com                    =20
FAX & Answer Phone 0871 242 8094
E-mail Accounts:=A0=20
=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0=A0 kemlo@tiscali.co.uk=A0or=20
             kemlo1@btinternet.com=A0
Disclaimer: The information contained in this message and/or any
attachments(s) may be of a private and confidential nature, and is
intended solely for the attention of the addressee. If you have received
this message in error or feel you should not have been the intended
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=A0
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-----Original Message-----
From: histonet-admin@lists.utsouthwestern.edu
[mailto:histonet-admin@lists.utsouthwestern.edu] On Behalf Of Margaret
Gondo
Sent: 15 September 2003 22:29
To: histonet@pathology.swmed.edu
Subject: [Histonet] eye morphology advice

Hi All -

I have a grad student who is interested in preserving the=20
morphology of a monkey eyeball once it is removed.  Does anyone=20
have any suggestions?  Everything we have tried always results in=20
the posterior part of the eyeball caving in. =20



Thanks in advance,
Margaret


=20


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




--__--__--

Message: 11
Date: Tue, 16 Sep 2003 08:10:58 -0400 (EDT)
From: "Barbara Osborn" 
To: "Histonet" 
Subject: [Histonet] Re:  Methacarn/Carnoy's Storage


Tamara,

You can store methacarn or Carnoy's for up to a couple of years as long 
as you cap it very, very tightly to avoid evaporation of chloroform and 
alcohol.  So you can store a large batch of fixative and only take out 
what is needed each week.  After use, however, it must be discarded.


--__--__--

Message: 12
To: "Histonet" 
From: Jackie.O'Connor@abbott.com
Date: Tue, 16 Sep 2003 07:30:32 -0500
Subject: [Histonet] IHC on Blood Smears

This is a multipart message in MIME format.
--=_alternative 0044E0DC86256DA3_=
Content-Type: text/plain; charset="us-ascii"

A colleague of mine would like to stain murine blood smears for Bcl2. Does 
anyone have any experience doing IHC on blood smears which have been fixed 
in Methanol? 

Thanks

Jacqueline M. O'Connor HT(ASCP)
Abbott Laboratories
Global Pharmaceutical Research and Development
Discovery Chemotheraputics
847.938.4919
Jackie.O'Connor@abbott.com
--=_alternative 0044E0DC86256DA3_=
Content-Type: text/html; charset="us-ascii"



A colleague of mine would like to stain murine
blood smears for Bcl2.  Does anyone have any experience doing IHC on
blood smears which have been fixed in Methanol? 



Thanks



Jacqueline M. O'Connor HT(ASCP)

Abbott Laboratories

Global Pharmaceutical Research and Development

Discovery Chemotheraputics

847.938.4919

Jackie.O'Connor@abbott.com
--=_alternative 0044E0DC86256DA3_=--


--__--__--

Message: 13
Date: Tue, 16 Sep 2003 10:14:27 -0400
From: "Fred Underwood" 
To: 
Subject: Re: [Histonet] eye morphology advice

I think this was discussed in the past.  If my memory serves me right,
injecting the eye with a gelatin solution before processing will help
retain the shape.  A search of the archives may be worth while.

Fred

>>> "Margaret Gondo"  09/15/03 05:28PM >>>
Hi All -

I have a grad student who is interested in preserving the 
morphology of a monkey eyeball once it is removed.  Does anyone 
have any suggestions?  Everything we have tried always results in 
the posterior part of the eyeball caving in.  



Thanks in advance,
Margaret


 


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


--__--__--

Message: 14
Date: Tue, 16 Sep 2003 10:14:27 -0400
From: "Fred Underwood" 
To: 
Subject: Re: [Histonet] eye morphology advice

I think this was discussed in the past.  If my memory serves me right,
injecting the eye with a gelatin solution before processing will help
retain the shape.  A search of the archives may be worth while.

Fred

>>> "Margaret Gondo"  09/15/03 05:28PM >>>
Hi All -

I have a grad student who is interested in preserving the 
morphology of a monkey eyeball once it is removed.  Does anyone 
have any suggestions?  Everything we have tried always results in 
the posterior part of the eyeball caving in.  



Thanks in advance,
Margaret


 


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


--__--__--

Message: 15
Date: Tue, 16 Sep 2003 10:26:51 -0400
From: "Fred Underwood" 
To: 
Subject: Re: [Histonet] IHC on Blood Smears

If the smears are quite thick and bloody, you may want to use a more
dilute hydrogen peroxide solution (1% or so) for quenching.  The
vigorous reaction can strip material from the slide.  Prolonged buffer
wahes are a good idea as well.

Fred

>>>  09/16/03 08:30AM >>>
A colleague of mine would like to stain murine blood smears for Bcl2.
Does 
anyone have any experience doing IHC on blood smears which have been
fixed 
in Methanol? 

Thanks

Jacqueline M. O'Connor HT(ASCP)
Abbott Laboratories
Global Pharmaceutical Research and Development
Discovery Chemotheraputics
847.938.4919
Jackie.O'Connor@abbott.com


--__--__--

Message: 16
Date: Tue, 16 Sep 2003 15:48:43 +0100 (BST)
From: "Vincenzo La Manna" 
To: 
Subject: [Histonet] immunohistochemistry and frozen sections



Hi Histonetters,

I've been struggling for 4 months on my 5 microns
frozen sections trying to find estrogen receptors.
I've tried both immunoperoxidase kit from Vector
laboratories either indirect immunofluorecsence (FITC
Goat anti mouse Secondary Ab from Sigma) but I've not
yet obtained good results.
My primary Ab is a mouse monoclonal to bovine
Estrogen receptor from Cymbus biotechnology.
Sections come from tissue that is supposed to be a
non-target tissue (mid laminar region from cow's
hooves)blocking with 2% goat normal serum (sigma)
whash in tbs buffer
All different methods for antigen retrieval have been
tested but sections look poor regarding histologic
quality and I still have very big problems with non
specific binding an staining (indirect
immunofluorescence).
It looks like frozen sections are too tender to
efford the whole processing.
Every suggestion or reference is welcome,
thank U

La Manna Vincenzo
Department of Agriculture and Forestry, University of
Aberdeen, 581 King Street, AB24 5UA, Aberdeen , UK.
Telephone; 01224 274259
Fax; 01224 273731
e-mail v.lamanna@abdn.ac.uk





--__--__--

Message: 17
Date: Tue, 16 Sep 2003 09:29:40 -0600 (MDT)
From: Phillip Huff 
To: "Vincenzo La Manna" 
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] immunohistochemistry and frozen sections

First, I would recommend that you run some Western Blots to ensure that
your Antigen of interest is being expressed within your tissues. Once your
Western blots are positive, you can definetly say that your immunoreaction
is what needs to be optimized.

Second, have you applied your immunoreaction technique to a known positive
tissue source, that has also been sectioned at 5 um? It may be a good
confirmation that your tissue may need to be processed differently or cut
thicker.

Finally, if your end goal is immunolocalization and the immunohistochem is
not working, you may want to look into in situ hybridization. In your
tissue samples, the receptor may be quite sensitive to processing and you
may be losing a great deal of your antigen. I know very little about your
receptor, but does it have a high turnover rate? Perhaps it has been
degraded over time when frozen. Has the reaction been done in freshly
harvested tissues? Perhaps localizing the mRNA in the tissues may provide
you with the results you are looking for. Before doing this though, I
would confirm that your tissues express the mRNA of interest by doing some
RT-PCR. This will both confirm that your tissues have your target mRNA,
and if you use an internal housekeeping primer in the PCR, you can
determine the approximate copy number of your mRNA to help you in
determining how sensitive your in situ reaction must be.

Hope this helps you out,

Phil



> Hi Histonetters,
>
> I've been struggling for 4 months on my 5 microns
> frozen sections trying to find estrogen receptors.
> I've tried both immunoperoxidase kit from Vector
> laboratories either indirect immunofluorecsence (FITC
> Goat anti mouse Secondary Ab from Sigma) but I've not
> yet obtained good results.
> My primary Ab is a mouse monoclonal to bovine
> Estrogen receptor from Cymbus biotechnology.
> Sections come from tissue that is supposed to be a
> non-target tissue (mid laminar region from cow's
> hooves)blocking with 2% goat normal serum (sigma)
> whash in tbs buffer
> All different methods for antigen retrieval have been
> tested but sections look poor regarding histologic
> quality and I still have very big problems with non
> specific binding an staining (indirect
> immunofluorescence).
> It looks like frozen sections are too tender to
> efford the whole processing.
> Every suggestion or reference is welcome,
> thank U
>
> La Manna Vincenzo
> Department of Agriculture and Forestry, University of
> Aberdeen, 581 King Street, AB24 5UA, Aberdeen , UK.
> Telephone; 01224 274259
> Fax; 01224 273731
> e-mail v.lamanna@abdn.ac.uk
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



--__--__--

Message: 18
Date: Tue, 16 Sep 2003 08:31:16 -0700
From: "Tommy Vedilago" 
To: "Histonet (E-mail)" 
Subject: [Histonet] Talented Histotechs wanted

Hello Histonetters,

  If you have ever wanted to work for a lab that is truly a family =
atmosphere where your skills and abilities are concretely appreciated, I =
have just the job for you. I have 3 positions for Histotechs in Dallas, =
TX with a progressive lab that has been in place for 36 years now. They =
are offering outstanding pay, benefits, and atmosphere. They are also =
offering relocation and hire bonus. If this holds interest for you or if =
you know of someone in the job market, please give me a call at =
866-SYSTEM1.
Tommy Vedilago
System 1 Search
(864) 627-0012
(864) 627-0013 Fax


--__--__--

Message: 19
Date: Tue, 16 Sep 2003 16:58:35 +0100 (BST)
From: nick.kirk3@btopenworld.com
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] immunohistochemistry and frozen sections

If it's not an inpolite question, why are you trying to demonstrate estrogen
receptors on frozen tissue?
Is this absolutely neccessary as estrogen receptors are very easily
demonstrated on paraffin processed tissue, which maintains better morphology
and gets almost guaranteed results.

We use the DAKO 1D5 clone on FFPE tissue using Vector Unmasking fluid for
Antigen Retrieval and the DAKO ChemMate detection system and get excellent
results every time.

Nick Kirk
Histopathology
Hinchingbrooke Hospital
Huntingdon
England

>  from:    Vincenzo La Manna 
>  date:    Tue, 16 Sep 2003 15:48:43
>  to:      histonet@lists.utsouthwestern.edu
>  subject: Re: [Histonet] immunohistochemistry and frozen sections
> 
> 
> 
> Hi Histonetters,
> 
> I've been struggling for 4 months on my 5 microns
> frozen sections trying to find estrogen receptors.
> I've tried both immunoperoxidase kit from Vector
> laboratories either indirect immunofluorecsence (FITC
> Goat anti mouse Secondary Ab from Sigma) but I've not
> yet obtained good results.
> My primary Ab is a mouse monoclonal to bovine
> Estrogen receptor from Cymbus biotechnology.
> Sections come from tissue that is supposed to be a
> non-target tissue (mid laminar region from cow's
> hooves)blocking with 2% goat normal serum (sigma)
> whash in tbs buffer
> All different methods for antigen retrieval have been
> tested but sections look poor regarding histologic
> quality and I still have very big problems with non
> specific binding an staining (indirect
> immunofluorescence).
> It looks like frozen sections are too tender to
> efford the whole processing.
> Every suggestion or reference is welcome,
> thank U
> 
> La Manna Vincenzo
> Department of Agriculture and Forestry, University of
> Aberdeen, 581 King Street, AB24 5UA, Aberdeen , UK.
> Telephone; 01224 274259
> Fax; 01224 273731
> e-mail v.lamanna@abdn.ac.uk
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet




--__--__--

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