Matt:LabNotes/2015-10-12
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DARTFISH on BA8 Sections[edit]
- Try one sample with second fixation step after permeabilization with TX-100 and Pepsin
- If sample is good use to compare with Gwen's RNAscope images of BA8 with 4 genes: OLFM1, PDE1A, SCL17A7, RELN
Protocol[edit]
Day 1[edit]
- Prepare 2 plastic culture dishes with 17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and RNase Free sterilize, then rinse with copious amounts of MilliQ water
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- Take out BA8 sections (2 coverslips) from -80C and incubate on 50C hot plate for 3min
- Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X PBS by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- One glass cracked so only one sample left DO SECOND FIXATION
- Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with cold 1X PBS three times
- Add 500ul 4% PFA for 10min at RT and
- Wash with cold 1X PBS three times
- Prepare 2X 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
- 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 |
Justin's Batch 7 8/31/2015 Padlock Probes 329nM]] | 5 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
Day 3[edit]
- Wash with 1X PBS twice
- 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 |
- 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 and store in 1X PBS at 4C
Day 4[edit]
- Add 200ul 0.5uM FISGA_Adpt in 2X SSC + 30% Formamide pre-heated to 75C and incubate for 10min at RT
- Wash with 2X SSC twice
- Add 400ul 2X SSC for imaging
10-26-2015[edit]
- When taking look at dish, noticed it was starting to leak and tried to seal again with superglue
- In the process, cracked the glass and had to throw away
Results[edit]
- Second fixation definitely helped retain tissue integrity
- Noticed much less tissue being washed away
- However number of rolonies is very few per cell
Images[edit]
- Tissue was oriented so the long axis was parallel with the y axis and the wider end was pointed "north" and the skinny end was "south"
- Right edge (East) is very straight and from deeper layer of cortex
- Left edge (West) is curved and rougher and from superficial layer of cortex
- Pos 1 is in the middle near the right edge where I saw the most tissue left on glass (opaque white)
- Pos 2-5 goes from deep layer to superficial layer at skinny end of tissue "south"
Pos 1[edit]
- Random position near center of tissue
File:MAX 20151015 BA8 DARTFISH Pos1 Gain600.jpg.jpg File:MAX cropped 20151015 BA8 DARTFISH Pos1 Gain600.jpg
Pos 2[edit]
- Near deep side (layer 6?) of skinnier end of section
File:MAX 20151015 BA8 DARTFISH Pos2 Gain550.jpg
Pos 3[edit]
File:MAX 20151015 BA8 DARTFISH Pos3 Gain550.jpg
Pos 4[edit]
File:MAX 20151015 BA8 DARTFISH Pos4 Gain550.jpg
Pos 5[edit]
- Near superficial side (layer 1?) of skinnier end of section