Hosuk:LabNotes/2014-10-14

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Human Brain Tissue : fixing test[edit]

  • Try Human Brain Tissue fixed with 4% Formaldehyde at 4C for 12hr.


Procedure[edit]

  • 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
    1. M-MuLV Buffer 20uL
    2. 25mM dNTP 2uL
    3. 2mM a-dUTP 4uL
    4. 100uM random hexamer RT Primer 5uL
    5. Rnase Inhibitor 2uL
    6. 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.