E16.5 C57BL6 SCGB1A1, CALCA, 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% Goat serum/PBS-T, 2 hours at RT.
- For mouse anti –CALCA (CGRP) (C7113, Sigma) dilute 1:200 in blocking buffer, 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 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 , Goat Alexa Fluor 488 anti-mouse IgG1 (A21121, Lot# 1345050, for anti –CALCA (CGRP) ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, anti-SCGBA1A1) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2A (A21136, Lot# 1345055, ACTA2) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 a light sealed box @ 4oC.
Crown-to-Rump Length: 16.0 mm
Antigen Retrieval Solution
18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5
82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2