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Created 9/17/13 by GEK Mixed Diane and Yunβs Protocols Human Nuclei Isolation and NeuN Staining for FCM/FACS #Place brain samples in the cryostat for ~20-30 minutes prior to beginning #While working in the cryostat, isolate 0.15-0.5g of tissue using a razor blade in a weigh boat. Cut into smaller pieces in the weigh boat. #Place tissue into 2 ml of cold CMF (HBSS or PBS +2 mM EGTA) (Calcium Magnesium Free) β Cut into smaller pieces with either forceps or the razor blade. Once minced, let sit for ~30 minutes. #Triturate the cells with 3 different bore size tips. (Cut them with a razor blade - ~4mm and 2 mm, and normal p1000 tip). Cells should be close to a single cell solution. #Pre-wet filter with PBSE. Filter suspension, wash with 2 mls of PBSE. #Spin down 5 mins at 1700 RPM #Aspirate supernatant, add 5 mls of PBSE+1% NP40 to lyse the cells. Add slowly while vortexing gently. Sit for 10 mins on ice with vortexing every 2 mins. #Spin down 5 mins 1700 RPM and aspirate supernatant. #Add 100ul PBSE to resuspend pellet. Add 4 mls of 70% (cold) ETOH and sit 30 mins (for sequencing work). Add 4 mls of 2-4% PFA for other applications. Skip this step for transcriptome work. #Spin down 5 mins at 1700 RPM, Aspirate, Wash in PBSE, Spin, Aspirate. #Resuspend in Blocking Buffer for 15-45 mins. #Add Primary Antibody (NeuN) and RNase for 20 mins. (or over night). #Without spinning, add ~3 volumes of PBSE, Spin, Aspirate, Wash, Spin, Aspirate. #Add Secondary antibody + DNA dye. If there is time leave ~1 hr. If time sensitive β leave in for sorting.
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