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2 Year-Old Human Lung Confocal Imaging Donor D022 SYP, SOX2, and TUBB3

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

Pryhuber_Lab

Immunofluorescence: SYPSOX2, and TUBB3

 

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for SYPSOX2, and TUBB3 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% Donkey serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-SYP (RB-1461-P, lot# 1461P1712D, Thermo Fisher) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) dilute 1:100, for mouse anti-