Hosuk:LabNotes/2014-10-14
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Human Brain Tissue : fixing test
- Try Human Brain Tissue fixed with 4% Formaldehyde at 4C for 12hr.
Procedure
- Fix 4% paraformaldehyde in 1x DEPC-PBS at 4C for 12 hr.
- Wash 2x in DEPC-PBE for 5 minutes each
- Glue and attach glass to MatTek dish
- Permeabilize in 0.25% Triton X-100 in DEPC-treated H2O for 15 min
- Wash 3 times in DEPC-H2O
- Add 200 ul 0.1% pepsin in 0.1 N HCl for 15 min at 37C
- Wash with 2 ml PBS three times to inactivate pepsin
- Add RT mix (Hexamer, +a-dUTP)
- DEPC-H2O 157uL
- M-MuLV Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- 100uM random hexamer RT Primer 5uL
- Rnase Inhibitor 2uL
- M-MuLV reverse transcriptase 10 uL
- Incubate the specimen with the reaction mixture for 15 min at 4°C, then transfer to 37°C for overnight.
Decoding PGP1F - Agi26k 20gap, 0gap, 0gap with Suppressor
- Decoding PGP1F with different Padlock Probes
- Dish1 : Agi26k_20gap
- Dish2 : Agi26k_0gap
- Dish3 : Agi26k_0gap with Suppressor
- Matt made 2nd rolonies
- Decode all 3 dishes at one time