E16.5 C57BL6 SOX9, phospho-Hist3H3, and ACTA2 Confocal Imaging

Immunofluorescence SOX9, pHISH3 and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL/6J E16.5 lungs for SOX9, pHISH3 and ACTA2 with antigen retrieval.

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 for 15 min at 96^oC. *We usually do a series of three runs 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% Donkey serum/PBS-T, 2 hours at RT.
10. For Rabbit anti-SOX9 (AB-5535, Lot # 2167153, Millipore) dilute 1:100, For goat anti p-Hist3H3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 4% Donkey serum/PBS-T. Spin for 5 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, Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1387792, for anti-SOX9), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-p-Hist3H3) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1366514, anti-ACTA2) at 1:200, 4% Donkey serum/PBS-T and 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 15 min.
5. Wash in PBS-T 3X, 5 min.
6. Rinse slides in 0.1M PB, 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, 24 X 50 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.14.24.4.19 E16.5

Gender: Male

Crown-to-Rump Length: 16.0 mm

Weight=0.592g

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