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

Immunofluorescence- LAMP-1, ABCA3, and SFTPC

Purpose: Stain slides of 5µm paraffin sections of human lung containing an ABCA3 homozygous missense mutation for  LAMP-1 and ABCA3 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% Donkey serum/PBS-T, 2 hours at RT.
10. For mouse anti-LAMP-1 (H4A3, Developmental Studies Hybridoma Bank) dilute 1:100 in blocking buffer, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) dilute 1:100, For Rabbit SFTPC (RB 458 in House, BL 3-23-11) dilute 1:100. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4^oC.

Day 2

1. Rinse slides in PBS-T 3X, 5 min.
2. Donkey Alexa Fluor 633 anti-mouse IgG1 (A21121, 1345050, LAMP-1) at 1:200, in blocking buffer Goat Alexa Fluor 568 anti-mouse pig IgG2B (A21144, Lot# 1584300, for anti-ABCA3) at 1:200, Donkey Alexa Fluor 647 anti-rabbit IgG (A31573, for anti-SFTPC),. 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

Tissue Used:
LMH-16-D036-RLL-12C1.21
Age: 8-years-old
Gender: Male

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