[Histonet] how to fix floating sections?
cbass <@t> wfubmc.edu
Tue Nov 9 15:51:02 CST 2010
I have a bunch of rat brains that I have frozen in isopentane and stored at –80. I'd like to collect 300 micron sections on a cryostat and collect tissue punches for RNA isolation. I would then like to take the remaining tissue and stain. There are a few ways of doing this, what would work the best is to take two 50 micron section, followed by a 300 micron, followed by two more 50, and go through the brain serially. I will then stain the 50 micron sections with nissl or thionin, so that the structures are easily identified and then use these to direct the tissue punches. The second 50 micron sections would be reserved for GFP IHC. Is this feasible? Here are some direct questions:
1. how would I store the 300 micron sections?
2. Any suggestions/protocols for staining the 50 micron sections for structure? Which stain would you use?
3. Can I post-fix the 50 micron sections? How about the 300 micron?
One reason I want to use floating sections for the nissl/thionin is that I can mount these very flat, no wrinkles and I'm very comfortable working with floating sections. I don't want to risk precious tissue trying to optimize this. One methodology recommended to me is to freeze a formalin solution in a 24 well plate, put the section on top flat and let them thaw together at room temperature or 4 degrees. This gives the tissue a nice, slow fixation.
Any other suggestions? How about working with the RNA? I'm thinking of using RNAlater-ice for example. Does anyone know if this screws up native fluorescence of GFP?
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