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==FISSEQ Protocol== ===Summary=== *Try without hydrogel, hopefully no degradation *Try 3 samples with 3 different pepsin incubation times #1 min #5 min #10 min ===Day 1=== #Prepare 3 plastic culture dishes with 10mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Make 4% PFA in 1X PBS #*10ml 16% PFA + 4ml 10X PBS + 26ml H2O (made 8-3-2016) #Take out mouse brain section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Attach coverslip to bottom of petri dish with ARcare 90106 double sided adhesive [[File:20160817_MouseBrain_Sample1.JPG|250px]] [[File:20160817_MouseBrain_Sample2.JPG|250px]] [[File:20160817_MouseBrain_Sample3.JPG|250px]] #Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Add 100ul 0.1% Pepsin in 0.1N HCl and incubate for X min at 37C #*6ul 5%Pepsin + 15ul 2N HCl + 279ul H2O #*HAD TO USE DIFFERENT PEPSIN ALIQUOT THAN NORMAL 1% #*When adding 5% pepsin to 0.1N HCl it was very white/cloudy almost like precipitate #*Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 10min #Wash with nf-1X PBS three times '''and check for degradation''' *Took Sample 1 out at 2min and saw that almost all tissue had fallen away *Took Sample 2 and 3 out right away (<3min) and the tissue was all gone *CONCLUSION: The pepsin concentration that Hosuk used over a year ago is way too high for these brain sections. Go back to 0.01% range for pepsin *Need to make new pepsin from powder and aliquot <!-- #Prepare 400ul Reverse Transcription Mix '''on ice''' and add ~133ul to each dish {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||159 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM RT primer||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~15hr at 37C ===Day 2=== #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C #Wash with nf-H2O twice -->
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