Hi all,
I am using IHC to look at various neuronal markers in zebra finch brain tissue. Finch brains are perfused with PBS and then 4% PFA and placed in a sucrose solution overnight. I then cryosection the tissue at 14 microns and place the tissue on super frost slides. Unfortunately, once I actually run the IHC experiments, the tissue disintegrates after a couple of washes with 0.3% TBS in Tx100. While cryosectioning, I place the brain on OTC to help with the sectioning but do nothing else to do the tissue. What could be causing tissue disintegration this early on in the procedure?
Thanks everyone.
Hi,
I would wash the sections with 0.3% Triton X-100 in 1X PBS instead of TBS. The Triton X-100 concentration may be too strong and cause the disintegration of your tissue so you could decrease it to 0.1% instead of 0.3%. Make sure the washes are not too long as well. If this doesn't work, I would recommend to simply cut thicker sections (~20µm).
You can also check this paper: Article A Simple Method for Immunohistochemical Staining of Zebrafis...
They recommend a sodium borohydride treatment.
Good luck!
Hi
Happy new year
Regarding to your IHC question, please answer this question:
Did you use any protocol to enhance attachment in your slides? I mean an specific gelatin or some thing else.
You did not mention this part so
Hi,
The same Suggestion, Make Triton 0.05% to 0.01 % (1 h blocking), then only wash with PBS (no detergent).
Also, check your fixation step, Usually, an Overnight post-fixation (4% PFA) can help, then, a sequential sucrose gradient get the job done (10% -> 20% -> 30%).
Best,
Emmanuel
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