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===Tissue Prep (~30 minutes)=== #Fixation ##Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice) ##Remove slides from -80C and immediately immerse in pre-chilled fixative for '''15 minutes''' #Dehydration ##Incubate slides in 50% EtOH for 5 minutes at RT ##Incubate slides in 70% EtOH for 5 minutes at RT ##Incubate slides in 100% EtOH for 5 minutes at RT ##Incubate slides in fresh 100% EtOH for 5 minutes at RT ##Air dry on a paper towel for 5 minutes at RT #Draw a hydrophobic barrier 2-4 times around each section with barrier pen; Let the barrier dry completely (~1 minute) #Turn on oven and set temperature to 40C; wet humidifying paper #Pretreatment ##Place slides on slide rack, add Pretreat 4 to entirely cover each section ##Incubate for 30 minutes at RT ###This time may be considerably shorter for different tissues, recommend 5 minutes for BA8 ###Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat ##Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS ##Wash slides in 1X PBS by moving rack up and down 3-5 times; repeat with fresh 1X PBS
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