Matt:LabNotes/2015-10-12

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Revision as of 23:05, 12 October 2015 by >Mzcai (→‎Day 1)
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DARTFISH on BA8 Sections

  • Try one sample with second fixation step after permeabilization with TX-100 and Pepsin
  • If sample is good use to compare with Gwen's RNAscope images of BA8 with 4 genes: OLFM1, PDE1A, SCL17A7, RELN

Protocol

Day 1

  1. Prepare 2 plastic culture dishes with 17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and RNase Free sterilize, then rinse with copious amounts of MilliQ water
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 sections (2 coverslips) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X PBS by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with cold 1X PBS three times
  11. To second fixation sample: Add 500ul 4% PFA for 10min at RT and then wash with cold 1X PBS three times
  12. Prepare 2X Reverse Transcription Mix on ice and add
Components Volume
H2O 159
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C