SPAM-LOW: [Histonet] Problem with liver fixation

[Patsy Ruegg]

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


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Itai Moshe
Sent: Sunday, September 05, 2010 6:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: [Histonet] Problem with liver fixation

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
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