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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

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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).
  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

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

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of SFTPC, ABCA3, and ACTA2

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of SFTPC, ABCA3, and ACTA2

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

Pryhuber_Lab

Immunofluorescence- SFTPC, ABCA3, and ACTA2

 

Purpose:  Stain slides of 5µm paraffin sections of human lung from the HTC for SFTPC, ABCA3, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC 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. Briefly equilibrate slides in Antigen Retrieval Buffer.
  4. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  5. 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).
  6. Microwave according to instructions on microwave.
  7. Cool on countertop, 15 min.
  8. Rinse with dH2O
  9. 1X PBS, 5 min.
  10. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  11. For Rabbit SFTPC (RB 458 in House, BL 3-23-11) dilute 1:500, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) 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 pig IgG2B (A21144, Lot# 1584300, for anti-ABCA3) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 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.
  10. Store slides in light-sealed box at 4oC.

 

Tissue Used:
LMH-D078-LLL-12B5.22
Gender: Female
Age: 7 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

By |2018-11-29T14:07:39+00:00November 29th, 2018|Categories: 07 Year-Old, Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments
Read More

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B 18B3

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B 18B3

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

Pryhuber_Lab

Immunofluorescence-KRT14, SOX2 and MUC5B

 

Purpose:  Immunofluorescence of 7µm frozen sections of lungs provided by HTC for KRT14, SOX2 and MUC5B 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 mouse anti-KRT14 (AB7800, Abcam) dilute 1:100, For mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) dilute 1:100, for rabbit anti-MUC5B (SC-20119, Lot # B1003 Santa Cruz) Dilute 1:100 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-mouse IgG3 (A21151, Lot# 1563682,  for anti-KRT14) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Goat Alexa Fluor 633 anti-rabbit IgG (A21070, Lot # 1668691, anti-MUC5B) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Rinse in PBS-T 5X
  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 in light-sealed box at 4oC.

 

 

 

Tissue Used:
2018-11-05_LMH-D078-RLL-18B3.13
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

By |2018-11-14T13:33:03+00:00November 14th, 2018|Categories: 07 Year-Old, Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments
Read More

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of KRT14, SOX2, and MUC5B

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

Pryhuber_Lab

Immunofluorescence-KRT14, SOX2 and MUC5B

 

Purpose:  Immunofluorescence of 7µm frozen sections of lungs provided by HTC for KRT14, SOX2 and MUC5B 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 mouse anti-KRT14 (AB7800, Abcam) dilute 1:100, For mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) dilute 1:100, for rabbit anti-MUC5B (SC-20119, Lot # B1003 Santa Cruz) Dilute 1:100 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-mouse IgG3 (A21151, Lot# 1563682,  for anti-KRT14) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Goat Alexa Fluor 633 anti-rabbit IgG (A21070, Lot # 1668691, anti-MUC5B) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Rinse in PBS-T 5X
  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 in light-sealed box at 4oC.

 

 

 

Tissue Used:
2018-11-05_LMH-D078-LLL-12B5.20
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

By |2018-11-13T17:48:24+00:00November 6th, 2018|Categories: 07 Year-Old, Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments
Read More

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2

7 Year-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of FN1, ABCA3, and ACTA2

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

Pryhuber_Lab

Immunofluorescence- FN1, ABCA3, and ACTA2

 

 

Purpose:  Stain slides of 7µm frozen sections of 3 year-old human lung provided by the HTC for FN1, ABCA3, 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% Donkey serum/PBS-T, 2 hours at RT.
  10. For rabbit anti-FN1 (AB2413, ABCAM, anti-Fibronectin), for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) 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, donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, anti-FN1), Goat Alexa Fluor 568 anti-mouse pig IgG2B (A21144, Lot# 1584300, for anti-ABCA3) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, 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.
  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:    

LMH-D078-LLL-13B4.18
Gender: Female
Age: 7 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

By |2018-10-12T18:14:30+00:00September 19th, 2018|Categories: 7 Year-Old, Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments
Read More

7 Years-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of SOX9, SOX2, and ACTA2

7 Years-Old Human Lung with Asthma Immunofluorescence and Confocal Imaging Donor D078 Showing Expression of SOX9, SOX2, and ACTA2

Immunofluorescence: SOX9, SOX2, and ACTA2 

 

Purpose:  Stain slides of 5µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC 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 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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) 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 , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti-SOX9) 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, ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 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-D078-RLL-18B3.16_SOX9_SOX2_ACTA2

Gender: Female

Age: 7 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

By |2018-09-13T16:49:46+00:00September 13th, 2018|Categories: Asthma Confocal Imaging, Human Lung Disease Confocal Imaging|0 Comments
Read More
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