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Author Topic: IHC on Frozen OCT cryosections with Acetone fixation Problems  (Read 2545 times)

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Offline LPatrick

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Hi everyone,  I am having a problem with my acetone fixation of 5uM cryosections of fresh tissue snap frozen in OCT.

The sections look fine when I cut them, and they look fine after fixing them in acetone, but after I wash them in buffer after drying off the acetone the tissues blow up.  They have excessive cell lysis, the lymphoid cells are almost completely destroyed and I have a long string effect of tissue damage, (looks like teased cotton-candy).   

My procedure is: Cut slides 5 uM, dry 1 hour in hood.  Fix 10 min in Fresh 4C 100% acetone 99.9% grade, dry 1 hour.  Wash 3x5 min in DPBS pH 7.4 No tween. Block for 30 minutes in 0.3% H202 in DPBS.  Wash 3x5 minutes in DPBS.  Block 30 minutes in 2% NGS in DPBS.  (I have been running hemotoxylin tests where i do an overnight incubation of 2% NGS in DPBS to simulate the conditions of my primary antibody incubation.

then I wash the slides in DPBS and Tap H20 and stain with hemotoxylin.

I am testing a 5 minutes incubation in 95% Etoh right after sectioning, with a 1 hour dry, then acetone fixing.

 I don't want to have to do this because we lost epitope signal when doing a methanol fixation instead of acetone.

Any advice appreciated.

Patrick