8 Month-Old Human Lung Confocal Imaging BPD Donor D086 Showing Expressions of HOPX, AGER, and ACTA2
Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center
- 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- HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, For goat anti-AGER (AF1145, R&D Systems) 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-HOPX), ), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-AGER), 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 D