Confocal Imaging for C57BL6
E16.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
- For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
- Briefly equilibrate slides in Antigen Retrieval Buffer.
- Antigen retrieval, pH 6.0 (times will vary according to microwave).
- 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96oC. *We usually do a series of three runs (6-7 minutes each run) to equal the time/temp because of evaporation (refill coplin jars with dH2O).
- Microwave according to instructions on microwave.
- Cool on countertop, 15 min.
- Rinse with dH2O
- 1X PBS, 5 min.
- Block in 4% Donkey serum/PBS-T, 2 hours at RT.
- For rabbit anti- NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For goat anti- FOXF1 dilute 1:100, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4oC.
- Rinse slides in PBS-T 3X, 5 min.
- Apply secondary antibody Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti- NKX2.1), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, anti-ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
- Rinse in PBS-T 3X, 5 min.
- Dilute DAPI 1:2000 and apply to slides for 10 min.
- Wash in PBS-T 3X, 5 min.
- Rinse slides in 0.1M PB or TB, 3X, 5 min.
- Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
- Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
- Allow Prolong Gold to cure overnight at room temp in light sealed box.
- Store slides in light sealed box @ 4oC