Difference between revisions of "Gelatin Embedding"
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(Created page with "== Gelatin Embedding == #Fixation in 1% MEMFA (1m FA, 4ml 10x MEM, 35 ml H2O) #Wash 3x for 10’ each in 1x PBS #Infiltrate tissue with 10% Sucrose in 1x PBS @ 4°C for 1 hou...") |
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#Fixation in 1% MEMFA (1m FA, 4ml 10x MEM, 35 ml H2O) | #Fixation in 1% MEMFA (1m FA, 4ml 10x MEM, 35 ml H2O) | ||
Latest revision as of 13:17, 26 July 2018
- Fixation in 1% MEMFA (1m FA, 4ml 10x MEM, 35 ml H2O)
- Wash 3x for 10’ each in 1x PBS
- Infiltrate tissue with 10% Sucrose in 1x PBS @ 4°C for 1 hour on the shaker until tissue sinks down
- Infiltrate tissue with 20% Sucrose in 1x PBS O/N @ 4°C on the shaker
- Incubate tissue in 3.5% gelatin in 20% Sucrose in 1x PBS O/N @ 37°C (Water bath)
i. 3.5g Gelatin in 100ml 20% Sucrose/1x PBS
6. Incubate tissue in 7.5% gelatin in 20% Sucrose in 1x PBS for at least 30’ @ 37°C (Water bath) until tissue will sink down
i. 7.5g Gelatin in 100ml 20% Sucrose/1x PBS
7. Embed tissue in 7.5% Gelatin in 20% Sucrose/1x PBS in the plastic mould
8. Freeze on dry ice
Cryostat:
- Object: -30°C
- Knife: -25°C