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Problem with liver fixation


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#1 megashock

megashock

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Posted 05 September 2010 - 04:22 AM

Hi,
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 in 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 will be better ?

Thank you all very much in advance




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