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 | 20 |
Ampligase Buffer | 10 |
Justin's Batch 5 7/22/2015 Padlock Probes 320nM]] | 26.3 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate 1hr at 37C
- Move to 60C as oven slowly decreases to 55C and held for another ~20hr