Postnatal Day 10 C57BL6 SOX2, NKX2.1, and ACTA2 Confocal Imaging
Immunofluorescence- SOX2, NKX2.1, and ACTA2
Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND10 lungs for SOX2, NKX2.1, and ACTA2 with antigen retrieval.
- 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% Goat serum/PBS-T, 2 hours at RT.
- For rabbit anti-SOX2 (R1236, BL 5/7/09, Seven Hills) dilute 1:200. For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-SOX2) 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, anti-ACTA2) at 1:200, in blocking buffer. Spin