Matt:LabNotes/2016-6-29
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FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9[edit]
- Used worst section from 1/19/16 BA8 patient 1568 from Yun
- These coverslips have only Vectabond (-NH2 primary amines) and no Bind-Silane
- 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[edit]
Day 1[edit]
- 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
Results[edit]
File:20160630 GelTissueSwellTest 1.JPG File:20160630 GelTissueSwellTest 2.JPG
- Next morning the gels were still stuck to glass
- No significant swelling
- Vertical length of gel was ~17mm when cast (no image but that is the length of the mold) and is ~17mm after overnight incubation in Tris
- Suggests that the gel is covalently bound to glass
Conclusion[edit]
- Making gel with Acrydite/Amine linker and BS(PEG)9 is viable solution to covalently bond polyacrylamide gel to Vectabond treated glass and tissue sections