Postnatal Day 03 C57BL6 SCGB1A1, NKX2.1, and TUBA1A Confocal Imaging
Immunofluorescence- NKX2.1, SCGB1A1, TUBA1A
Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND03 NKX2.1, SCGB1A1, and TUBA1A with antigen retrieval.
- Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. 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.
- Briefly equilibrate slides in Antigen Retrieval Buffer.
- 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96oC. *We usually do a series of three runs 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 Rabbit NKX2.1 (R1231) dilute 1:1000. For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti-TUBA1A1 (T7451, Sigma) dilute 1:1000 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-rabbit IgG (A11034, Lot# 1298480, for TTF-1), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1549801, Acetylated α-Tubulin) 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 15 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.
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