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. 1 min
  2. 5 min
  3. 10 min

Day 1[edit]

  1. Prepare 3 plastic culture dishes with 10mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O (made 8-3-2016)
  3. Take out mouse brain section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. 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

  1. Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
  2. Wash with cold nf-H2O three times and check for degradation
  3. 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
  4. 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