From:Luz Cardenas

This is the protocol we are using in the Lab. I hope this help.
Do I need to use always antigen retrieval/unmasking for cryostat sections?.
Thank you in advance.
1.Perfusion with: 0.9% saline and 4% PFA in 0.1M PBS-->storage in last solution overnight.
2. Place in 20% Sucrose until sink
3. Put Brain in crymold with OCT--> bath in 2 methyl butane-->chunk in dry ice with oct and placed the brain-->put in cryostat and wait around  1 h ( at 20oC)
4. Sectioning 20uM-->used gelatine slide-->air dry overnight.
5. Do Vector ABC kit-->blocking solutions
rinse in PBS 5', 2 times
6. Primary-chat ab (chemicon), overnight-->chat 1:3000
rinse PBS 5' , 2 times
7. Do vector ABC kit--> secondary 30'
rinse (=)
8.Do vector ABC kit-->detector 30'
rinse (=)
9. DAB (sigma): used sigma protocol. like 5' or until color desired ?
rinse in PBS
10. dry and coverslip

Luz Cardenas, MD
Post-doctoral Research
Department of Anatomy and Neurobiology
University of Tennessee Health Science Center

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