4-Month-Old Enhanced Resolution Confocal Imaging Donor D075 for SFTPB and ABCA3

Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center

Immunofluorescence- SFTPC, SFTPB , and ABCA3

Purpose:  Stain slides of 5µm paraffin sections of 4 month-old postnatal human lung from HTC SFTPC, SFTPB, andABCA3 with antigen retrieval.

Day 1

1. Paraffin sections are placed at 60^oC 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. Antigen retrieval, pH 6.0 (times will vary according to microwave).
4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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 SFTPC (RB 458 in House, BL 3-23-11, pro-SFTPC) dilute 1:500, for guinea pig anti-SFTPB (GP20, in house, mature SFTPB) dilute 1:100, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) dilute 1:100 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4^oC.

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-SFTPB), Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1366514, anti-ABCA3)  at 1:200, in blocking buffer. Spin down in µfuge for 20 min, apply to tissue and incubated at room temperature for 1 hour.
3. Wash 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.
10. Store slides in light sealed box @ 4^oC.