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===Day 1=== #Prepare 1 plastic culture dishes with hole, tweezers, large plastic dish, slide gasket, and 50C hot plate #*EtOH and RNaseZap sterilize and rinse with nf-H2O #Made 40ml 4% PFA at RT #*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O #Take out BA8 section (1 glass slide1 and 1 coverslip1) from -80C and incubate on 50C hot plate for 3min #Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C #Wash twice with 2X SSPE by submerging #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive #Aspirate any liquid and attach gaskets to glass slides #Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT #Wash with nuclease-free H2O three times #Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C #*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O #Wash with nuclease-free PBS three times #Prepare 3X Reverse Transcription Mix '''on ice''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||477 |- | 10X M-MuLV Buffer||60 |- | 25mM dNTP||6 |- | 4mM aa-dUTP||6 |- | 100uM N9 or 100uM FISSEQ_RT||15 |- | RNase Inhibitor||6 |- | M-MuLV RTase||30 |- | Total||600 |} #Incubate 10min at 4C and then ~13hr at 37C #*Put slide in 50ml tube with wet towel and para film around cap #*Parafilm dish and put in humidified bag with mouse E7.5
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