Rat hippocampus homogenized in RIPA buffer with P/P inhibitors. BCA. Heated 70 degrees for 10 min. Run on 4-12% bis-tris gel with MOPS buffer. 7 Minute xfer with trans-blot turbo. Occludin 1:500 overnight with 1:10,000 800 CW Li-Cor secondary.
If you use brain tissue as such, you'll probably have a fairly low concentration, since the capillaries with the occludin constitutes less than 1 % of brain volume. There has however been reported occludin in the neurons of the hippocampus, is that what you're looking for? Do you have a positive control for you primary AB? I would suggest to increase the pAB or isolate the capillaries (if possible, you'd have to pool quite a bit of tissue). This wouldn't work if you're going for neuronal expression, though...
I changed primary antibody and concentration and had instant success.
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