I am looking for an optimal method to perform a Ki67 and C/EBPα staining on frozen sections from adipose tissue.
My current protocol exists of the following steps and gives disappointing results:
1. Dry up after taking out the sections from the freezer (at room temperature for 10 minutes).
2. Post fixation with PFA 4% (at room temperature for 30 minutes). PBS washing (at room temperature for 2x15 minutes).
3. Methanol 100% (at room temperature for 10 minutes). Drying for 10 minutes. PBS washing (at room temperature for 2x15 minutes).
4. Blocking with goat serum 10% in PBS (at room temperature for 60 minutes).
5. Primary antibody (at 4 degrees, overnight). I use
- C/EBPα (D56F10) XP rabbit monoclonal antibody (from Cell Signaling, #8178)
- anti-mouse/rat Ki-67 purified antibody (from eBioscience, 14-5698)
Followed by PBS washing (at room temperature for 2x15 minutes).
6. Secondary antibody (at room temperature, 1 hour). I use goat anti-rabbit IgG-Alexa 594 for the C/EBPα and goat anti-rat IgG-Alexa 594 for the Ki-67. Followed by PBS washing (at room temperature for 2x15 minutes).
7. Mounting with Vectashield Mounting Medium with DAPI.
The DAPI-positivity is normal, but I cannot demonstrate the presence of Ki-67 and C/EBPα in my sections. Since Ki-67 and C/EBPα are nuclear markers, I have tried to increase the cell membrane's permeability with proteinase K instead of methanol 100%, but this did not improve our results.
Additional information: before cryosectioning, the adipose tissue has been fixed with PFA 4% for 7 days (with PFA 4% changed daily) and has undergone cryoprotection (sucrose 6% - 12% - 18% - 24%, each 3 hours, and then 30% during the night). The sections have a thickness of 6 and 12 micrometer and are sectioned following the Kawamoto method (cryofilm).
Does anybody have experience with Ki-67 and/or C/EBP-α staining on frozen adipose tissue sections? Or other ideas to increase permeability of the membrane to stain nuclear proteins or transcription factors?
Thank you
Hi dear, I think this article would be good
Article Ultrarapid Ki-67 immunostaining in frozen section interpreta...
Ki 67 is intranuclear antigen, you have to do permeabilization of the cells using triton X, tis may help .
good luck
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