Single Cell RNA-Seq and Gene Info

Immunofluorescence- SFTPC, NKX2.1, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57Bl/6 E18.5 lungs for NKX2.1, SFTPC, 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 for 15 min at 90oC. *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% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-SFTPC (RB458, BL 8/24/11, in house) dilute 1:500, for guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:200, for mouse anti- ACTA2 (α Smooth muscle actin, 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-SFTPC), 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, 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.14.27.6.62 E18.5

Gender: Male

Crown-to-Rump Length: 21.0 mm

Weight=1.32g

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