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===Day 1=== #Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O #Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole) #*When peeling off scotch tape from coverslip (that was used to tape coverslips to glass slides), it peeled off a clear film as well... #*What kind of clear film would develop on coverslip at -80C? #*Use Double-sided tape: Adhesives Research IS-8458-19 #Wash tissue section twice using cold nf-SSPE for 5min each #*A8 leaked so used 200 Gap filling glue #Add 0.25% TX-100 in nf-2XSSPE for 5min at RT #Wash with cold nf-H2O twice #Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C #Wash with 2ml of cold nf-PBS three times #Prepare 2X Reverse Transcription Mix '''on ice''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||157 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM FISSEQ_RT||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~18hr at 37C #*Parafilm each dish and then put in plastic bag with wet tissue
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