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2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expression of FN1, FOXF1, and ABCA3

2 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D022 Showing Expression of FN1, FOXF1, and ABCA3

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

Pryhuber_Lab

Immunofluorescence- FN1FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC  for FN1FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3(WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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 @ 4oC.

Tissue Used:   
2018-12-13_LMH_D022-LLL-4A4.26
Age: 2 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

Related Experiments

Recent Experiments

By |2018-12-14T15:25:24+00:00December 14th, 2018|Categories: 02 Year-Old, Homo sapiens Confocal Imaging|0 Comments

14 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D072 Showing Expression of FN1, FOXF1, and ABCA3

14 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D072 Showing Expression of FN1, FOXF1, and ABCA3

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

Pryhuber_Lab

Immunofluorescence- FN1FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC  for FN1FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3(WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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 @ 4oC.

Tissue Used:   
2018-12-11_LMH_D072-RLL-14B2.20
Age: 14 Months
Gender: Female

 

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

Recent Experiments

By |2018-12-12T14:11:35+00:00December 12th, 2018|Categories: 14 Month-Old, Homo sapiens Confocal Imaging|0 Comments

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, FOXF1, and ABCA3

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, FOXF1, and ABCA3

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

Pryhuber_Lab

Immunofluorescence- FN1FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC  for FN1FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3(WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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 @ 4oC.

Tissue Used:   
2018-12-10_LMH_D078-LLL-13B4.19
Age: 7 Years
Gender: Female

 

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

Recent Experiments

By |2018-12-12T13:48:16+00:00December 12th, 2018|Categories: Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments

Human Lung Confocal Imaging MC17.5H.A3.7 showing expressions of FN1, FOXF1 and ABCA3

Human Lung Confocal Imaging MC17.5H.A3.7 showing expressions of FN1, FOXF1 and ABCA3

Immunofluorescence- FN1FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC and microlithiasis human lung for FN1FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3(WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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.
  1. Store slides in light sealed box @ 4o

 

Tissue Used: 
2018-12-05_LMH_MC17-5H.A3.7 

 

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

Recent Experiments

By |2018-12-11T13:12:32+00:00December 11th, 2018|Categories: Human Lung Disease Confocal Imaging, Primary Alveolar Microlithiasis|0 Comments

Human Lung Confocal Imaging MC17.5H.B1.8 showing expressions of FN1, FOXF1 and ABCA3

Human Lung Confocal Imaging MC17.5H.B1.8 showing expressions of FN1, FOXF1 and ABCA3

Immunofluorescence- FN1, FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC and microlithiasis human lung for FN1, FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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.
  1. Store slides in light sealed box @ 4o

 

Tissue Used:
2018-12-04_LMH_MC17-5H.B1.8   

 

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

Recent Experiments

By |2018-12-07T17:54:44+00:00December 6th, 2018|Categories: Human Lung Disease Confocal Imaging, Primary Alveolar Microlithiasis|0 Comments

3 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expression of FN1, FOXF1, and ABCA3

3 Year-Old Human Lung Immunofluorescence and Confocal Imaging Donor D032 Showing Expression of FN1, FOXF1, and ABCA3

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

Pryhuber_Lab

Immunofluorescence- FN1, FOXF1 , and ABCA3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC  for FN1, FOXF1 , and ABCA3

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-FN1 (AB2413, ABCAM, anti-Fibronectin) dilute 1:100, For goat anti- FOXF1 dilute 1:100, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) dilute 1:100  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, anti-FN1) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-FOXF1), at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, 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).