Hi everyone,
I have a problem since 1-2 months to optimize a laminin staining in a fixed adult mouse brain. I went through the literature however I couldn't solve the problem:
I have perfect staining around the cortex, however the staining intensity strongly decreases the more deep I go to the deeper layer of the brain.
I tried to fix for 4h, 12h, 24h in PFA after IC perfusion. And I tried different permeabilization techniques and the use of 37°C for primary antibody incubation.
Attached you can see an image...i would be extremely greatful, if someone could help me out.
Thank you a lot!
Hi Ruslan, are you working with mounted sections and if it is so, how thick they are? If they are thicker than 20 µm I would recommand to proceed the IHC on free floating sections. Your image might show an artifact which appears when the sections not quiet tied stick on the slide. The antibody does not only stain the surface, it also can get behind the section and this leads to a more intensiv staining in the border area of the sectons. Hope, this will help you.
Dear Ruslan Rust, did you find a solution to this staining issue?
I have the same pattern of staining in mouse (adult) brain, after free-floating staining of 50µm vibratome-cut sections. I consider increasing Triton concentration (right now I am using 0.04% v/v) or leaving my sections in the primary for a longer duration (36-48h instead of 12-18h now), but I would be happy to hear any advice from you.
Thank you very much!
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