Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2

Grow the cells on either 8µm chamber slide or 48-well plate. (1x105cells/well)

•     Coating and plating densities can be found in the differentiation kit
  

Aspirate medium and rinse with 1x PBS

Fixation: Cover cells with warm 4% PFA and fix for 20 min at RT.

•     Cover the vessel and place in the hood
  

Rinse 3X in 1x PBS for 5 min each at the lab bench

Blocking and permeabilize for 30 min at RT with Blocking Buffer

1º antibody incubation dilute in 1/3 blocking buffer + 1x PBS and incubate overnight in the cold room

-20º (box labelled antibodies for iPSCs)

•     FOXA2 Rb à (1:250)
  

•     OTX2 Mus IgG à (1:250)
  

o Add 4µl of antibody per 1mL of prepared antibody solution

Rinse three times in 1x PBS for 5 min each

2º antibody incubation for 1hr at RT in the dark

•     A11008 Alexa Flour 488 goat anti-rabbit IgG (HHH+L) 1:400 in PBS
  

•     A10036 Alexa Flour546 donkey anti-mouse IgG (H+L) 1:400 in PBS
  

o Add 2.5µL of antibody per 1mL of prepared antibody solution

*Combine these two antibodies in one tube. Ex. If you were making 1.6mL (for 8 wells) you would add 4µL of each antibody

Rinse 3X in 1X PBS for 5 min each

Incubate with DAPI at (1µg/mL) for 10min at RT (Stock is 1mg/mL which is 1000x)

a. Add 1µl of Stock DAPI to 1mL of prepared solution.

11. Rinse 2X in 1X PBS and image.