Postnatal Day 1 C57BL6 EMCN, AGER, and ACTA2 Confocal Imaging
Day 1
- 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% Donkey serum/PBS-T, 2 hours at RT.
- For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, For rat anti-AGER (MAB 1179-500, RND Systems) dilute 1:100, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4oC.
Day 2
- Rinse slides in PBS-T 3X, 5 min.
- Apply secondary antibody, Donkey Alexa Fluor 488 anti-goat IgG (A11055, Lot# 1605893, for anti-EMCN)at 1:200, Donkey Alexa Fluor 568 anti-rat IgG (AB175708 for anti-AGER) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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 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 light sealed box @ 4oC
Tissue Used: LMM.15.66A.6.64
PND01 C57BL6
Gender: Female
Weight= 1.14g
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 dH2 O
pH 6.0
