Matt:LabNotes/2016-1-27
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FISSEQ on Mouse Embryo Test 2
- Previous test using sections of fixed embryo
- This time try fresh frozen mouse embryo sections
Embryo Section Info
Protocol
- Previously did 0.01% Pepsin for 10min at RT and had too much tissue loss
- Try 0.01% Pepsin for 1-2min at 37C
Day 1
- Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Use 1 week old 4% PFA at RT
- Take out fresh frozen mouse embryo sections 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
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
- 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
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Parafilm each dish and then put in plastic bag with wet tissue