Matt:LabNotes/2016-1-20
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DARTFISH on BA8 Sections[edit]
- Last time imaged with 20X objective didn't decode enough rolonies
- 90 sec 0.01% pepsin incubation worked well
- 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]
- Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Prepare fresh 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
- Take out 2 best BA8 sections (2 furthest back) from -80C and dry 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
- 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[edit]
- Wash with 1X PBS once
- 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
- 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
- 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 |
- Add mix to sample and incubate 30min at 37C
- Move to 60C as oven slowly decreases to 55C and held for another ~18hr
Day 3[edit]
- 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
- 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
- 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 |
- Wash with 1X PBS once
- 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
Check Rolony[edit]
- Add 0.5uM FISGA_Adpt in 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image (saved in 1-26-2015)
- Strip with 80% formamide preheated to 75C
- 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