Matt:LabNotes/2015-10-29
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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
- Prepare 1 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Use 40ml 4% PFA at RT made by Dan on same day
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
- Take out last BA8 section (~1 month ago) from -80C and incubate on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Slightly more stripes in gray matter
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times and check for degradation
- Still looks good File:20151029 BA8 DARTFISH PostPepsin.JPG
- 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 |
- Incubate 10min at 4C and then ~18hr at 37C
Day 2
- Wash with 1X PBS once
- Gray matter looks slightly thinner than before RT... but could be lighting
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- At the maximum width, width = 8mm. The border from 7-8mm is very thin
- Store in 1X PBS 2 days at 4C
Day 3 11/2/2015
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
- 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 |
- Add mix to sample and incubate 30min at 37C
- Move to 60C as oven slowly decreases to 55C and held for another ~20hr
Day 4
- Wash with 1X PBS once
- 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
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- 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
- Wash with 1X PBS once
- Gray matter is still there (I can see the thin stripes of tissue) but very thin
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- Wash with 1X PBS twice
- Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
- Wash sample with 10mM HEPES
- Add beads to sample and incubate 5min at RT
- Wash sample with 10mM HEPES twice
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1X Tris pH 8.0 for 30min at RT
- 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
- Pos2: 51.49, 37.99
- Pos3: 52.71, 37.99
- edge of tissue where it starts to look complete/whole
- 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
- 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