Matt:LabNotes/2016-8-17
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Mouse Brain FISSEQ test[edit]
Previous mouse brain FISSEQ tries[edit]
2nd try
3rd try
4th try
7th try
9th try
4X BF Image of mouse brain sections from Yun
Sample Info[edit]
- Received 10 x 10um thick sections of mouse brain on Vectabond and Bind-Silane treated coverglass
- Each section is half of coronal section
- Fresh frozen tissue, cryostat into 10um sections on vectabond coverslips, dry for 30min at -20C in cryostat chamber
- Box up and freeze at -80C
- Coverslip attached to glass slide by a drop of frozen water
FISSEQ Protocol[edit]
Summary[edit]
- Try without hydrogel, hopefully no degradation
- Try 3 samples with 3 different pepsin incubation times
- 1 min
- 5 min
- 10 min
Day 1[edit]
- 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 File:20160817 MouseBrain Sample2.JPG File:20160817 MouseBrain Sample3.JPG
- 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