Matt:LabNotes/2015-10-29: Difference between revisions

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**Superficial edge with ~50% of cells/tissue still intact
**Superficial edge with ~50% of cells/tissue still intact
**z thickness <7um
**z thickness <7um
[[File:20151029_AllRolonies_BF_Pos1.jpg|450px]]
*Pos2: 51.49, 37.99
*Pos2: 51.49, 37.99
*Pos3: 52.71, 37.99  
*Pos3: 52.71, 37.99  
**edge of tissue where it starts to look complete/whole
**edge of tissue where it starts to look complete/whole
[[File:20151029_AllRolonies_BF_Pos3.jpg|450px]]
*Pos4: 53.50, 37.99
*Pos4: 53.50, 37.99
*Pos5: 54.49, 37.99
*Pos5: 54.49, 37.99
Line 131: Line 133:
**Deep edge of tissue
**Deep edge of tissue
**z thickness >15um
**z thickness >15um
[[File:20151029_AllRolonies_BF_Pos8.jpg|450px]]


*At approx the widest point, tissue is ~8mm wide
*At approx the widest point, tissue is ~8mm wide

Revision as of 07:00, 8 November 2015

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
    • Gray matter looks slightly thinner than before RT... but could be lighting
  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. Store in 1X PBS 2 days at 4C

Day 3 11/2/2015

  1. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  2. Wash with nf-H2O twice
  3. 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 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
  1. Add mix to sample and incubate 30min at 37C
  2. Move to 60C as oven slowly decreases to 55C and held for another ~20hr

Day 4

  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
  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

Day 5

  1. Wash with 1X PBS once
    • Gray matter is still there (I can see the thin stripes of tissue) but very thin
  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
  6. Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
  7. Wash sample with 10mM HEPES
  8. Add beads to sample and incubate 5min at RT
  9. Wash sample with 10mM HEPES twice
  10. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  11. Wash with 1X PBS twice
  12. Add 1X Tris pH 8.0 for 30min at RT
  13. Wash with 1X PBS twice

Imaging Results

Positions Imaged

  • Pos1: 50.54, 37.99
    • Superficial edge with ~50% of cells/tissue still intact
    • z thickness <7um

File:20151029 AllRolonies BF Pos1.jpg

  • Pos2: 51.49, 37.99
  • Pos3: 52.71, 37.99
    • edge of tissue where it starts to look complete/whole

File:20151029 AllRolonies BF Pos3.jpg

  • Pos4: 53.50, 37.99
  • Pos5: 54.49, 37.99
  • Pos6: 55.58, 37.99
  • Pos7: 56.73, 37.99
  • Pos8: 58.32, 37.99
    • Deep edge of tissue
    • z thickness >15um

File:20151029 AllRolonies BF Pos8.jpg


  • At approx the widest point, tissue is ~8mm wide
  • The superficial 2mm are partially degraded but still has rolonies in the nuclei that are intact

Conclusion

  • 90sec pepsin digestion and very careful gentle washes helped keep much more of the tissue
  • 37C incubation for 30min at the start of Ampligase reaction helps a LOT
  • 20+hr RCA reaction led to larger rolonies, but since density isn't as high not an issue
    • Might be better for 20X 0.75NA objective
  • For decoding to validate with RNAscope (where tile image is 5.6mm wide starting from superficial edge) I will only be able to compare the 3.6mm after 2mm superficial layers
    • Check RNAscope quantification of genes to make sure there is changes in gene expression in those layers