E16.5 C57Bl6 SOX2, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- SOX2, NKX2.1, and ACTA2 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 E16.5 lungs for SOX2, NKX2.1, and ACTA2 with antigen retrieval.

Day 1

1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
2. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by 3X in 100% ETOH for 3 minutes each, 95% ETOH for 3 minutes each, and 70% ETOH for 3 minutes each. Slides are then placed in 1X PBS for 5 minutes to completely rehydrate tissue.
3. Briefly equilibrate slides in Antigen Retrieval Buffer.
4. 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).
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 Rabbit anit-SOX2 (R1236, BL 5/7/09, Seven Hills) dilute 1:200, For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer and incubate on tissue overnight @ 4oC.

Day 2

1. Rinse slides in PBS-T 3X, 5 min.
2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-SOX2), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1366514, for anti-ACTA2 ) at 1:200, in blocking buffer. Spin down antibody cocktail in a µfuge for 10 min 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 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.

Tissue Used:

LMM.14.24.8.7 E16.5

Gender: Male

Crown-to-Rump Length: 16.5 mm

Weight=0.644g

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

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