1) Prepare one solution of 1N NaOH and 1mL of 10x PBS. Keep them on ice.
2) Put the amount collagen needed and pour it in an ependorf tube. Do it in the hood with sterile forceps. Keep the collagen on ice
3) Here are the proportion for the different components of the gel:
- 9 parts collagen in 1 part 10x PBS
- 23 micro liters of 1N NaOH in 1 mL of collagenase
For example:
- 500mL of collagenase
- 55 mL of PBS
- 11.5 mL of NaOH
4) First, mix PBS and NaOH. Then, add collagen. Keep the gel on ice.
5) Take the plate with the EBs
6) Pour the solution in a falcon tube
7) You can either spin down the tube at 700tr/min for 2 minutes or let the EBs sink. The last solution is better.
8) Suck the medium and wash the cells with PBS
9) Same step as 7)
10) Make a small mold and put it in a small petri dish
11) Take the gel and pour it in the falcon tube where the cells are
12) Take the whole gel with the EBs and pour them in the mold.
13) Leave the mold in the petri dish in the fridge for about 15 minutes so the EBs sink in the gel
14) Then, put the petri dish in the incubator for about 30 minutes
15) After 30 minutes in the incubator, remove the mold
16) Add about 7mL of 3.2% PFA to fix the cells
17) Leave the dish in the fridge for 1 to 2 hours
18) Remove the PFA and add PBS for 1 hour
19) Remove the PBS and add 70%EtOH overnight
20) Now, we can embed the sample from step 4 of the embedding protocol (protocol 9).