E16.5 C57Bl6 SFTPC, phospho-Hist3H3, 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 for 15 min at 96oC. *We usually do a series of three runs 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-SFTPC (RB458, BL 8/24/11, in house) dilute 1:1000. For goat anti-p-Hist3H3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, and incubate on tissue overnight @ 4oC.
- Rinse slides in PBS-T 3X, 5 min.
- Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti-SFTPC), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-p-Hist3H3) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1549801, for 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 15 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 l