E18.5 C57BL6 SCGB1A1, CALCA, and ACTA2 Confocal Imaging

Day 1

1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
2. Briefly equilibrate slides in Antigen Retrieval Buffer.
3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
5. Microwave according to instructions on microwave.
6. Cool on countertop, 15 min.
7. Rinse with dH₂O
8. 1X PBS, 5 min.
9. Block in 4% Goat serum/PBS-T, 2 hours at RT.
10. 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 @ 4^oC.

Day 2

1. Rinse slides in PBS-T 3X, 5 min.
2. 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.
3. Rinse in PBS-T 3X, 5 min.
4. Dilute DAPI 1:2000 and apply to slides for 10 min.
5. Wash in PBS-T 3X, 5 min.
6. Rinse slides in 0.1M PB or TB, 3X, 5 min.
7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
9. Allow Prolong Gold to cure overnight at room temp in light sealed box.
10. Store slides in a light sealed box  @ 4^oC.

Tissue Used:

LMM.14.49.5.82 E18.5

Gender: Male

Crown-to-Rump Length: 22.0 mm

Weight=1.17g

Appendix

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

1L dH₂O

pH 6.0