Gelatin Embedding

From Tanaka Wiki
Jump to: navigation, search
  1. Fixation in 1% MEMFA (1m FA, 4ml 10x MEM, 35 ml H2O)
  2. Wash 3x for 10’ each in 1x PBS
  3. Infiltrate tissue with 10% Sucrose in 1x PBS @ 4°C for 1 hour on the shaker until tissue sinks down
  4. Infiltrate tissue with 20% Sucrose in 1x PBS O/N @ 4°C on the shaker
  5. 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