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Author Topic: 50um thick sections  (Read 3009 times)

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Offline dcarter

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50um thick sections
« on: February 04, 2010, 05:10:48 PM »
Hello
I have been given this project of staining 50um thick fixed-frozen monkey brain sections. The PI is looking at the nigral tracts for stereology. My problem is, the IHC is not penetrating throughout the sections. I am free-float staining, I have increased the time of the primary antibody from1 day to 2 days, I have increase the timing of the secondary, and the ABC to 1.5 hrs each. She is saying that the staining only penetrate around 7um on both sides of the sections,  there is no staining in the middle.  I pretreat with 0.1Triz and triton x-100. Please help me

50um thick sections
« on: February 04, 2010, 05:10:48 PM »

Offline CanuckPhD

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Re: 50um thick sections
« Reply #1 on: February 05, 2010, 02:24:30 AM »
One thing you can try is to use 0.3% Triton instead of 0.1%. I am assuming that you have your sections on a shaker during the entire procedure.

Thick sections aren't my area of expertise (I usually only do 20 um) but often the ABC complexes can be very large, which may hinder their diffusion into the middle of the thick section. Fluorescent tagged secondaries might be a better bet. Or maybe try some of the biotin free systems that are now available.

Re: 50um thick sections
« Reply #1 on: February 05, 2010, 02:24:30 AM »