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==Linnarsson Lab Protocol== [[Media:33_Protocol_SC_smFISH_Stellaris.pdf|Linnarsson lab protocol]] #Prepare fresh paraformaldehyde 4% and keep at RT #Fix sections with 4% paraformaldehyde for 15 minutes at RT #Rinse sections twice with RT PBS #Permeabilization-may use: ##Ethanol (70% EtOH, >90 minute incuabation at 4C) ##PBS-Tween 20 ##PBS-TritonX-100 ##PBS-Igepal ##Methanol (usually the worst) #Rinse sections twice with RT PBS #Prepare the wash buffer and let it sit at RT #Incubate sections in wash buffer for 10 minutes #Warm hybridization buffer and warm it up to 37C #Prepare hybridization reaction: ##hybridization buffer-100 uL; probe 2-10 uL #Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc. '''Day 2''' #Warm up wash buffer and wash buffer with DAPI to 37C #Rinse with wash buffer #Add fresh was buffer and incubate for 30 min at 37C #Replace with fresh wash buffer with DAPI and incubate for 30 min at 37C #Wash 2 times with warm 2X SSC #Mount the slide
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