Matt:LabNotes/2015-10-12

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DARTFISH on BA8 Sections[edit]

  • 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[edit]

Day 1[edit]

  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
    • One glass cracked so only one sample left DO SECOND FIXATION
  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. Add 500ul 4% PFA for 10min at RT and
  12. Wash with cold 1X PBS three times
  13. 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

Day 2[edit]

  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. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. Prepare Ampligase mix on ice
    • Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
Component Volume
DEPC-H2O 20
Ampligase Buffer 10
Justin's Batch 5 7/22/2015 Padlock Probes 320nM]] 26.3
Justin's Batch 7 8/31/2015 Padlock Probes 329nM]] 5
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
  1. Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr

Day 3[edit]

  1. Wash with 1X PBS twice
  2. To DARTFISH slide add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. Add RCA mix and incubate 10.5hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200
  1. Wash with 1X PBS once
  2. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice and store in 1X PBS at 4C

Day 4[edit]

  1. Add 200ul 0.5uM FISGA_Adpt in 2X SSC + 30% Formamide pre-heated to 75C and incubate for 10min at RT
  2. Wash with 2X SSC twice
  3. Add 400ul 2X SSC for imaging

10-26-2015[edit]

  • When taking look at dish, noticed it was starting to leak and tried to seal again with superglue
  • In the process, cracked the glass and had to throw away

Results[edit]

  • Second fixation definitely helped retain tissue integrity
    • Noticed much less tissue being washed away
  • However number of rolonies is very few per cell

Images[edit]

  • Tissue was oriented so the long axis was parallel with the y axis and the wider end was pointed "north" and the skinny end was "south"
  • Right edge (East) is very straight and from deeper layer of cortex
  • Left edge (West) is curved and rougher and from superficial layer of cortex
  • Pos 1 is in the middle near the right edge where I saw the most tissue left on glass (opaque white)
  • Pos 2-5 goes from deep layer to superficial layer at skinny end of tissue "south"

Pos 1[edit]

  • Random position near center of tissue

File:MAX 20151015 BA8 DARTFISH Pos1 Gain600.jpg.jpg File:MAX cropped 20151015 BA8 DARTFISH Pos1 Gain600.jpg

Pos 2[edit]

  • Near deep side (layer 6?) of skinnier end of section

File:MAX 20151015 BA8 DARTFISH Pos2 Gain550.jpg

Pos 3[edit]

File:MAX 20151015 BA8 DARTFISH Pos3 Gain550.jpg

Pos 4[edit]

File:MAX 20151015 BA8 DARTFISH Pos4 Gain550.jpg

Pos 5[edit]

  • Near superficial side (layer 1?) of skinnier end of section

File:MAX 20151015 BA8 DARTFISH Pos5 Gain550.jpg