Matt:LabNotes/2016-6-15: Difference between revisions
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>Mzcai m (→Day 1) |
>Mzcai m (→Day 1) |
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Line 18: | Line 18: | ||
#Wash twice using cold nf-SSPE | #Wash twice using cold nf-SSPE | ||
#Add TX-100 in nf-2XSSPE for 5min at RT | #Add TX-100 in nf-2XSSPE for 5min at RT | ||
#*0.5% for 10, 15, 20, 20min at RT while | #*0.5% for 10, 15, 20, 20min at RT while on orbital shaker | ||
#Wash with cold nf-H2O twice | #Wash with cold nf-H2O twice | ||
#Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 5min at 37C | #Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 5min at 37C |
Revision as of 23:10, 15 June 2016
FISSEQ whole mount embryo
- Objective:
- Test multiple harsher permeabilization conditions
- Embed in thin (~50um) PA hydrogel for imaging (FISH and stripping)
- Last time realized no signal past outermost layer of cells
- Hoping more permeabilization will allow rolony reaction to occur throughout embryo
Procedure
- Embryos are fresh and sitting in 1X PBS in 200ul PCR tube
Day 1
- Sterilize bench and tweezers with EtOH, RNaseZap
- Make fresh 4% PFA in PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
- Add Argon gas and seal with parafilm to keep it fresh longer
- Aspirate PBS from tube and add 200ul 4% PFA
- Incubate at 37C for 15min
- Wash twice using cold nf-SSPE
- Add TX-100 in nf-2XSSPE for 5min at RT
- 0.5% for 10, 15, 20, 20min at RT while on orbital shaker
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 5min at 37C
- Wash with 2ml nf-PBS three times
- Prepare 2X Reverse Transcription Mix on ice
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 ~16hr at 37C