[Histonet] Questions about IHC in Frozen Sections

[Lewis, Patrick]

Hi Everyone.

I am trying to troubleshoot  my IHC on frozen sections.

My sections are human tonsil at 7 uM. On charged Superfrost slides.

They are stored at -80 after drying for 1 hour.

When I use them for IHC, I take them out of the -80 and let them air dry for 1 hour before placing them in cold acetone for 30 minutes to fix.

Question:
If I place them directly in H20 or TBST pH 8.0 after fixation, will that cause cell lysis?

Should I dry the slides after acetone fixation before washing them?

If so, for how long?

My problem seems to be that the tissue is getting digested on the slide, I am trying to trouble shoot which step is causing my tissues to disintegrate.

So far I have tried thicker sections 10, 15 uM (That made the problem worse, I am consider going back to 4 uM sections)

I also Changed the concentration of H2O2 for my H202 block from 3% to 0.3%,
(In my next IHC attempt I will try to examine the slide at each step to see if I can see loss of integrity)

Also in my next attempt I plan to eliminate any H20 washes and dry the slide post acetone fixation before washing in TBST.

Also I plan to decrease the amount of Tween20 in my Wash buffer from 0.2% to 0.02%.

Any advice would be helpful.

Patrick.




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