Matt:LabNotes/2015-10-12: Difference between revisions
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>Mzcai m (→Day 1) |
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#Wash twice with cold 1X PBS by submerging | #Wash twice with cold 1X PBS by submerging | ||
#Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive | #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 | #Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT | ||
#Wash with cold nf-H2O three times | #Wash with cold nf-H2O three times | ||
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#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | ||
#Wash with cold 1X PBS three times | #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 | #Prepare 2X Reverse Transcription Mix '''on ice''' and add | ||
{| {{table}} | {| {{table}} |
Revision as of 01:39, 13 October 2015
DARTFISH on BA8 Sections
- 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
Day 1
- 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