Matt:LabNotes/2016-2-23
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DARTFISH on BA8 Sections with PA gel
- Used worst section from 1/19/16 BA8 patient 1568 from Yun
- Gel mix and protocol is same as this successful experiment
- 4% Formaldehyde is one month old and has been sitting at RT
- From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf>
- "We recommend that 10% buffered formalin solutions be used no longer than
3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5."
Protocol
Day 1
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Used 4% PFA from 1/20/2016
- Take out worst BA8 sections (furthest front) 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 100ul gel casting mix filtered and degassed
- Cover with plastic coverslips cut and glued together such that it leaves 0.5mm space for gel height
- FORGOT TO DO THIS SO GEL WON'T BE FLAT ON TOP
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with cold 1X SSPE
- Mostly did not polymerize
- Only a ~1cm diameter circle of gel polymerized in center
- I will continue with FISSEQ protocol to see whether can generate as many rolonies in gel area as non-gel area
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- 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 ~15hr at 37C
Day 2
- 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
- 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 FISSEQ mix on ice
- Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component | Volume |
DEPC-H2O | 640 |
CircLigase Buffer 10X | 100 |
MnCl2 50mM | 50 |
Betaine 5M | 200 |
CircLigase II (100U/ul) | 10 |
Total | 1000 |
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Wash with 2X SSC + 30% formamide for 10min at 60C
- wash with 2X SSC, 1X SSC, 1X PBS once each
- Add 200ul RCA mix and incubate for 15hr 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 3
- 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
- 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
- Imaged 1-26-2016
- 63X with 0.3um step size
- 2048x2048
- Gain = 600 Offset = 1%
Sample A
White matter edge
- Edge of tissue on vertical side
File:MAX BA8 A white edge.jpgFile:BA8 A white edge z24 ch01.jpg
2mm from edge
File:MAX BA8 A 2mm from edge.jpgFile:BA8 A 2mm from edge z18 ch01.jpg
4mm from edge
File:MAX BA8 A 4mm from edge.jpgFile:BA8 A 4mm from edge z21 ch01.jpg
6mm from edge
File:MAX BA8 A 6mm from edge.jpgFile:BA8 A 6mm from edge z15 ch01.jpg
Sample B
White matter edge
- Edge of tissue on vertical side
File:MAX BA8 B white edge.jpgFile:BA8 B white edge z15 ch01.jpg
2mm from edge
File:MAX BA8 B 2mm from edge.jpgFile:BA8 B 2mm from edge z15 ch01.jpg
4mm from edge
File:MAX BA8 B 4mm from edge.jpgFile:BA8 B 4mm from edge z15 ch01.jpg
Conclusion
- 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
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