[Histonet] Re: Problem with liver fixation

[Itai Moshe]

Thank's all,

I'm thinking that my IHC method might be O.K because it is working great
with other paraffin embeddedd tissues like diaphragm and various muscles,
and (sometimes) also in older liver tissues that i have found in our lab
(but i don't know how they were made).

I'm using antigen retrieval with 0.01% Citrate buffer, pH 6.0 heated in
microwave.
but I don't rule out that my IHC method is not optimal for liver tissues,
and that there might be a better one.

Does someone have a proven fixation, or IHC methods that worked with live
tissues ?

Thank you all very much

Itai M



2010/9/7 Andrea Marion <amario3 <@t> uic.edu>

> Hi Itai,
>
> Liver antigens tend to degrade rapidly, so immediate fixation is
> necessary. It sounds like you are getting the tissue into the solution
> quickly enough. Fixation occurs more slowly at 4 degrees than room
> temperature though - perhaps your antigen is degrading during this
> 'slower' fixation? Are you using a sufficient volume of fixative compared
> to tissue mass?
>
> Another concern is that some antigens require paraformaldehyde fixation
> (ie a 4% solution of buffered formaldehyde prepared directly from solid
> paraformaldehyde). Solutions of formaldehyde marketed as '37%
> formaldehyde' or formalin typically contain ~10% methanol added as a
> stabilizer, which can interfere with some antigens. For
> immunohistochemistry purposes, you may need to prepare your formaldehyde
> solution directly from paraformaldehye. See here for more discussion of
> formaldehyde solutions: http://publish.uwo.ca/~jkiernan/formglut.htm
>
> Finally, you list 'IHC protocol', but why are you sure your problem lies
> in the tissue fixation/processing and not the staining protocol? You
> probably need to do some form of antigen retrieval - what method are you
> using? What is your primary antibody dilution? Has your antibody been
> validated for use with IHC/IF? Many antibodies simply do not work with
> FFPE tissues. There are many other steps that could be causing trouble...
> Here is a good beginner's guide for IHC with FFPE tissues if you need:
> http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf
>
> As a side note, your processing steps may be a little long, but I am not
> an expert. We use only 30 minutes for each dehydration and clearing step,
> and 2 paraffin steps for one hour each with mouse tissue. Good luck!
>
> Andrea Marion
>
> amario3 &at& uic &dot& edu
> Graduate Student
> University of Illinois at Chicago
>
>
> ----
>
 Rene J Buesa <rjbuesa <@t> yahoo.com>  histonet <@t> lists.utsouthwestern.edu ‏,
Itai Moshe


Bouin's will not do better than what you describe. Are you using antigen
retrieval?
René J.
----
Patsy Ruegg <pruegg <@t> ihctech.net>
Itai Moshe itai.moshe <@t> mail.huji.ac.il ‏,

I would not fix at 4c, do it at rt.  Your fixative looks ok and processing,
actually for animal tissues I process for only 20 min a station not 60 min.,
animal tissues will process quicker because they have less fat, and you can
over process them so they become dryed out and hard.

About not getting a signal no matter what ab you use, there could be several
causes, are you using the proper antigen retrieval technique for that ab, is
your detection system matched to your primary antibody, on and on..........?

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org

---

Itai Moshe <itai.moshe <@t> mail.huji.ac.il>אל histonet <@t> lists.utsouthwestern.edu

Dear All,
I'm trying to use liver sections in paraffin blocks for IHC, but get no
signal at all, doesn't matter what antibody i'm using.

I'm using section about 3mm thick (the natural thickness of mouse liver) and
about 5-7mm Width and Length.

My fixation protocol is like this:
1) immediately after killing the mouse i'm putting the sections in a
fixation solution that is made from: 10ml formaldehyde 37%, 5ml PBSx20, 85ml
DDW  - pH 7, 24Hr at 4C.
2) ETOH 70%, ETOH 80%, ETOH 96%, ETOH 100% x2 - each for 1Hr at RT
3) Xylen x2 - each for 1Hr at RT.
4) Paraffin x3 - each at 60C for 1Hr.

Before doing IHC:
1) Xylen x2 - each for 10Min at RT
2) ETOH 100%x2, ETOH 96%, ETOH 80%, ETOH 70% - each for 2Min at RT.
3) IHC protocol...

Will Bouin's solution be better ?

Thank you all very much in advance

Itai M