Is it possible to permebilize frozen brain sections in order to help antibody access the intracellular epitopes of an antigen? Would treatment with detergents such as triton or NP-40 or Tween-20 work on frozen sections? Kindly help!
If you want permeate, you may need to perform first a mild fixation with PFA. If not the detergent will wash of the tissue from your slides. It can be done, at least with unfixed skin is my experience, but the fine-tuning could be rather technical. Good luck!
I tried a mild fixation using formalin. However, I was able to visualize only mild positives (no strong Immunofluorescence). Would doing it with 4% PFA make a difference? Or is it mandatory to fix the tissue primarily in 4% buffered formaldehyde for 24 h, followed by dehydration and paraffin embedding?
Detergent choice depend on
-the brain section size... Do you use superfrost plus slides for little section? The tissue wouldn't be wash if you use this type of slide.
-Specie of interest: Do you work on mouse, rat, human or other brain?
-Fixation: What fixation? If you use ethanol or methanol you don't have to permeabilize. PFA 4% or acetone, you can use triton x100 at 0,05% to 1% with the primary antibody or step before saturation.
-If the tissue is delicate or thin (
Hi VM
Thanks for your suggestions.
I work with human brain sections and these are
Saponin 0,05% would work on your section.
But it might not because the antigen of interest can be "masked" by formalin fixation.
I think you have to see the datasheet of your antibody to see if the antigen need to be unmasked with formalin fixation.
In order to avoid this problem on unfixed frozen sections i prefer to fix with aceton -20°C 10 min for section size 1cm2.
Kindly,
VM
Yes, I am trying out acetone fixation next since it does not require any permeabilization procedure after that. I hope it works!
Hi Christoph
I tried the IFT experiments with 10% and 4% formalin and permeabilzed with 0.5% Triton-X. However, the fluorescence intensity were greatly reduced. I tried also acetone fixation but in vain. So, I performed the IFT with EUROIMMUN OT's just as it is. Let me know if i can help you further!
Best
Ishu
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