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8 Year-Old Human Lung Confocal Imaging Donor D036 SFTPC, KI67, and ACTA2

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

Pryhuber_Lab

                                             Immunofluorescence- SFTPC, Ki-67and ACTA2

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC for SFTPC, Ki-67, and ACTA2 with antigen retrieval.

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  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 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).
  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) dilute 1:500, For mouse anti-Ki-67 (55603, BD Biosciences, Lot# 6251774)
    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

  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 568
    anti-mouse IgG1 (A21124, Lot# 1600878, for anti-Ki-67) 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 20 min, apply to tissue and incubated at room
    temperature for 1 hour.
  1. Rinse in PBS-T 3X, 5 min.
  2. Dilute DAPI 1:2000 and apply to slides for 15 min.
  3. Wash in PBS-T 3X, 5 min.
  4. Rinse slides in 0.1M PB, 3X, 5 min.
  5. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  6. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  7. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  8. Store slides in light sealed box @ 4o

 

Tissue Used:
LMH-16-D036-RLL-12C1.11
Age: 8 Years
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

 

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