Matt:LabNotes/2016-9-21
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DARTFISH on BA8 with SplintR in PA Gel[edit]
- Try 1 sample with polyacrylamide gel added after permeabilization
- Used 200nM padlock probe (twice the normal concentration)
- 5% Gel mix and protocol is same as this with Acryloyl-X
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
10mg/ml Acryloyl-X, SE in DMSO | 1 |
H2O | 39.75 |
5% TEMED | 1 |
5% APS | 1 |
Total | 50 |
Protocol[edit]
Day 1[edit]
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Made 4% PFA in 1X PBS on 8/31/2016
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out BA8 section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- 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
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 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 1X PBS
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash once with 1X PBS
- Prepare SplintR Mix
- Preheat Padlock Probes then snap cool
Component | Volume |
10X SplintR Buffer | 10 |
268nM | 80 |
H2O | 10 |
Total | 100 |
- Move sample to 60C and oven slowly decreases to 55C and held for ~24hr
Day 2[edit]
- Wash with 1X PBS twice preheated to 55C
- Sample was dry in center, unusual amount of condensation on lid. Image borders of tissue should be okay.
- Add SplintR Enzyme and incubate at 37C for 20min
- 20ul 10X Buffer + 6ul SplintR + 174ul H2O
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Add RCA mix and incubate at 30C overnight (~15hrs)
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 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
Imaging[edit]
- Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image (saved in 9-23-2016)
- Taken with zstep = 1um except 'Edge' with zstep = 0.3um
- 3 Images taken
- Middle - the area that dried up during padlock probe capture
- Edge - Close to superficial layer that most likely did not dry
- Between - Somewhere in between middle and edge
Results[edit]
Middle[edit]
Between[edit]
Edge[edit]
Conclusion[edit]
- Rolony density is higher than ever seen before with FISSEQ or DARTFISH
- Especially in 'edge' image, which is near superficial layer 1 of neocortex that normally has least amount of rolonies
- Specificity probably good because of nuclear enrichment of rolonies
- If non-specific then wouldn't see so much more in nucleus than anywhere else
- Decreased rolony density in middle is most likely due to dried up during padlock probe annealing and not indicative of poor results
- NEXT STEP: Decode this sample to prove real rolonies and then Order New Padlock Probes that is RevComp of TB12k_Apr2016