Matt:LabNotes/2016-8-18: Difference between revisions

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>Mzcai
(Created page with "=Mouse Brain FISSEQ test= *Repeat yesterday's experiment but with way less pepsin ==Previous mouse brain FISSEQ tries== [[Hosuk:LabNotes/2014-5-19...")
 
>Mzcai
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#Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
#Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
#Wash with cold nf-H2O three times '''and check for degradation'''
#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
#Add 100ul 0.01% Pepsin in 0.1N HCl and incubate for X min at 37C
#*30ul 1%Pepsin + 15ul 2N HCl + 255ul H2O
#*3ul 1%Pepsin + 15ul 2N HCl + 282ul H2O
#*Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 10min
#*Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 10min
#Wash with nf-1X PBS three times '''and check for degradation'''
#Wash with nf-1X PBS three times '''and check for degradation'''

Revision as of 22:59, 18 August 2016

Mouse Brain FISSEQ test

Previous mouse brain FISSEQ tries

2nd try
3rd try
4th try
7th try
9th try
4X BF Image of mouse brain sections from Yun

Sample Info

  • 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

Summary

  • 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

  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 fresh)
  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
  7. Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  9. Add 100ul 0.01% Pepsin in 0.1N HCl and incubate for X min at 37C
    • 3ul 1%Pepsin + 15ul 2N HCl + 282ul H2O
    • Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 10min
  10. Wash with nf-1X PBS three times and check for degradation
  11. Prepare 400ul Reverse Transcription Mix on ice and add ~133ul to each dish
Components 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
  1. Incubate 10min at 4C and then ~15hr at 37C