Matt:LabNotes/2015-10-29

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Revision as of 06:27, 30 October 2015 by >Mzcai (→‎Day 1)
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DARTFISH on BA8 Sections

  • Now I know the regions that have been degrading the most are the gray matter with cortical layers
    • Need this to compare with Gwen's tilescan images
  • Try 90sec 0.01% pepsin incubation and pay close attention to gray matter region for degradation

Protocol

Day 1

  1. Prepare 1 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Use 40ml 4% PFA at RT made by Dan on same day
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
  3. Take out last BA8 section (~1 month ago) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times and check for degradation
    • Slightly more stripes in gray matter
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nf-1X PBS three times and check for degradation
  11. Prepare 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

Day 2

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. Store in 1X PBS 2 days at 4C