Matt:LabNotes/2016-1-20

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DARTFISH on BA8 Sections[edit]

  • Picked up 9 sections from Yun today: "1/19/16 Matt & Yun Vectabonded coverslips BA8 patient 1568"
    • He said many of the sections had "shattering" near the superficial layers of the thicker end (The curved part of 'P')
    • The best sections are in the back of the box and the worst are in the front
      • The very worst had tissue wiped off one side of coverslip and then better section stuck to the other side

Protocol[edit]

Day 1[edit]

  1. Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Prepare fresh 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
  3. Take out 2 best BA8 sections (2 furthest back) from -80C and dry 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[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
    • At the maximum width, width = 8mm. The border from 7-8mm is very thin
  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
  • B9: 216nM
Component Volume
DEPC-H2O 17.43
Ampligase Buffer 10
Justin's Batch 8 7/22/2015 Padlock Probes 298nM]] 34
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
Component Volume
DEPC-H2O 5.43
Ampligase Buffer 10
Justin's Batch 9 7/22/2015 Padlock Probes 216nM]] 26
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
  1. Add mix to sample and incubate 30min at 37C
  2. Move to 60C as oven slowly decreases to 55C and held for another ~18hr

Day 3[edit]

  1. Wash with 1X PBS once
  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
    • Washed with 1X PBS another time and let sit in PBS for ~10min at RT because the RCA mix partially froze in the chilled tube holder so I had to remake the RCA mix
  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

Check Rolony[edit]

  1. Add 0.5uM FISGA_Adpt in 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
  4. Image (saved in 1-26-2015)
  5. Strip with 80% formamide preheated to 75C
  6. Wash with 1X PBS twice


Imaging Results[edit]

  • Imaged 1-26-2016
  • 63X with 0.3um step size
  • 2048x2048
  • Gain = 600 Offset = 1%

Sample A[edit]

White matter edge[edit]

  • Edge of tissue on vertical side

File:MAX BA8 A white edge.jpgFile:BA8 A white edge z24 ch01.jpg

2mm from edge[edit]

File:MAX BA8 A 2mm from edge.jpgFile:BA8 A 2mm from edge z18 ch01.jpg

4mm from edge[edit]

File:MAX BA8 A 4mm from edge.jpgFile:BA8 A 4mm from edge z21 ch01.jpg

6mm from edge[edit]

File:MAX BA8 A 6mm from edge.jpgFile:BA8 A 6mm from edge z15 ch01.jpg

Sample B[edit]

White matter edge[edit]

  • Edge of tissue on vertical side

File:MAX BA8 B white edge.jpgFile:BA8 B white edge z15 ch01.jpg

2mm from edge[edit]

File:MAX BA8 B 2mm from edge.jpgFile:BA8 B 2mm from edge z15 ch01.jpg

4mm from edge[edit]

File:MAX BA8 B 4mm from edge.jpgFile:BA8 B 4mm from edge z15 ch01.jpg

Conclusion[edit]

  • Even with this minimal pepsin digestion and careful washing there's too much tissue degradation
  • Problem is compounded by the fact that the most interesting area (grey matter with cortical layers) is also the most fragile
  • Need to try adding polyacrylamide gel layer over the tissue to prevent tissue degradation