Menu

13 Month-Old Human Lung Confocal Imaging Donor D083 SYP, SOX2, and TUBB3

13 Month-Old Human Lung Confocal Imaging Donor D083 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-TUBB3 (anti-tubulin β 3, cat. # 801201, Biolegend) dilute 1:200 in blocking buffer. Spin down in µfuge for 20 minutes and incubate on tissue overnight @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti-SYP) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, TUBB3) 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. 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.
  1. Store slides in light sealed box @ 4oC

 

 

Tissue Used:
LMH-16-D083-LLL-8A3.7
Gender: Male
Age: 13 Months

           

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

 
 
 
 
 

Related Experiments

3 Year-Old Human Lung Confocal Imaging Donor D053 SYP, SOX2, and TUBB3

3 Year-Old Human Lung Confocal Imaging Donor D053 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-TUBB3 (anti-tubulin β 3, cat. # 801201, Biolegend) dilute 1:200 in blocking buffer. Spin down in µfuge for 20 minutes and incubate on tissue overnight @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti-SYP) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, TUBB3) 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. 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.
  1. Store slides in light sealed box @ 4oC

 

 

Tissue Used:
LMH-16-D053-LLL-6B2.17
Gender: Male
Age: 3 Years

           

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

Related Experiments