Matt:LabNotes/2016-3-28
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DARTFISH on BA8 Sections with PA gel[edit]
- Last time including 4% PFA in hydrogel resulted in no rolonies
- Guessing the PFA cross-linked the gel too much so pore size was too small for diffusion
- Before that without 4% PFA the hydrogel swelled and also no rolonies
- Guessing this is because permeabilized AFTER gel polymerization
- This time do tissue permeabilization before gel polymerization
- Don't use 4% PFA in the hydrogel solution
- I don't think swelling will be too big an issue
- Used 5th best section from 1/19/16 BA8 patient 1568 from Yun
- 4% Formaldehyde for fixation is three weeks old and has been sitting at RT under argon gas
- 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 3/8/2016
- Take out second best remaining BA8 sections (5th from back) 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
- 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
- Add 75ul gel casting mix filtered and degassed
- 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
- Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height
- 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 cold 1X SSPE
- Edges did not polymerize but the grey matter is fully covered
- Prepare 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
Day 2[edit]
- There was almost no solution left on coverslip though there was around edges of dish
- Was it bumped, tilted, or moved by someone?
- Gel swollen and wrinkled as expected
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice