Matt:LabNotes/2016-6-29: Difference between revisions
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>Mzcai m (→Day 1) |
>Mzcai m (→Day 1) |
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#Aspirate non-polymerized gel and wash once with 1X PBS | #Aspirate non-polymerized gel and wash once with 1X PBS | ||
#Attach coverslip to bottom of petri dish | #Attach coverslip to bottom of petri dish | ||
#*COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL | |||
#*NEED TO BE MORE CAREFUL WHEN HANDLING | |||
*Continued by testing for swelling | |||
#Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight | |||
<!-- | |||
#Add 1M Tris and let sit at RT for 15min | #Add 1M Tris and let sit at RT for 15min | ||
#Wash once with cold 1X SSPE | #Wash once with cold 1X SSPE |
Revision as of 03:16, 30 June 2016
FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9
- Used worst section from 1/19/16 BA8 patient 1568 from Yun
- Gel mix and protocol is same as this successful experiment
- Except half the A/B mix to make 5%
- 4% Formaldehyde is two weeks old and has been sitting at RT
- From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf>
- "We recommend that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should
not be below 6.5."
Protocol
Day 1
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Used 4% PFA from 6/15/2016
- Take out worst BA8 section that is left 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
- 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 120um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 100ul gel casting mix filtered and degassed
- 5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9
- 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
- COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL
- NEED TO BE MORE CAREFUL WHEN HANDLING
- Continued by testing for swelling
- Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight