Postnatal Day 28 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
Immunofluorescence SFTPC, SCGB1A1, and ACTA2
Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND28 lung for SFTPC, SCGB1A1, 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-SFTPC (RB458, BL 8/24/11, in house) dilute 1:1000, For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, 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.