After taking 250micrometer thick rodent brain section (by vibratome in aCSF) and incubate them with 1mg/ml of PI for 20 mins and then 1microgram of DAPI for next 40 min ( with/without fixing the tissue with PFA), I found that the slices does not picking the stain and section were further lend to mince up sometimes, Kindly suggest.
For immunohistochemistry, I would recommend thinner brain sections if possible. We prepare brain sections 30-50 um thick. Antibody penetration is inversely correlated with tissue section thickness. While DAPI is much smaller molecule than antibodies, it will also not penetrate throughout a very thick section. To improve staining, use a tissue detergent (such as Triton X or Tween 20 in 0.1 to 0.5 % in your staining solution - PBS or TBS).
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