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8 Month-Old Human Lung Immunofluorescence and Confocal Imaging BPD Donor D086 Showing Expressions of AGER, PECAM1, and NKX2.1

8 Month-Old Human Lung Immunofluorescence and Confocal Imaging BPD Donor D086 Showing Expressions of AGER, PECAM1, and NKX2.1

 

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

Pryhuber_Lab

Immunofluorescence- AGER, PECAM1, NKX2.1

 

Purpose:  Stain slides of 7µm frozen sections of postnatal human lung from HTC for AGER, ABCA3, NKX2.1 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 goat anti-AGER (AF1145, R&D Systems) dilute 1:100, For mouse anti-PECAM1 (CD31 , BBA7, R&D Systems) dilute 1:100, For Rabbit anti-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:500 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  1. Apply secondary antibody , Donkey Alexa Fluor 488 anti-goat IgG (A11055, lot# 1687906, anti-AGER), Donkey Alexa Fluor 568 anti-mouse IgG (A10037, Lot# 1752099, anti-PECAM1 ) at 1:200, Donkey Alexa Fluor 647 anti-rabbit IgG (A31573, anti-1) at 1:200 in blocking buffer. Spin down in µfuge for 10 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 10 min.
  3. Wash in PBS-T 3X, 5 min.
  4. Rinse slides in 0.1M PB or TB, 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 a light-sealed box at 4oC.

 

Tissue Used:
LMH-17-D086-LLL-8A3.9
Age: 8 Months-old
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

By |2018-10-18T12:44:33+00:00April 20th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
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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: SYP, SOX2, and TUBB3

 

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for SYP, SOX2, 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

By |2018-11-12T16:22:18+00:00March 26th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
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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: SYP, SOX2, and TUBB3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for SYP, SOX2, 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

By |2018-11-12T16:23:39+00:00March 23rd, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

3 Year-Old Human Lung Confocal Imaging Donor D039 SYP, SOX2, and TUBB3

3 Year-Old Human Lung Confocal Imaging Donor D039 SYP, SOX2, and TUBB3

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

Pryhuber_Lab

Immunofluorescence: SYP, SOX2, and TUBB3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for SYP, SOX2, 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-D039-LLL-6A3.10
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

By |2018-11-12T16:58:31+00:00March 23rd, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

8 Month-Old Human Lung Confocal Imaging Donor D086 SYP, SOX2, and TUBB3

8 Month-Old Human Lung Confocal Imaging Donor D086 SYP, SOX2, and TUBB3

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

Pryhuber_Lab

Immunofluorescence: SYP, SOX2, and TUBB3

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for SYP, SOX2, 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-D086-LLL-8A3.22
Gender: Male
Age: 8 Months old

                       

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 |2018-11-12T18:09:30+00:00March 21st, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

3 Year-Old Human Lung Confocal Imaging Donor D015 FN1, FOXF1, and ACTA2

3 Year-Old Human Lung Confocal Imaging Donor D015 FN1, FOXF1, and ACTA2

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

Pryhuber_Lab

                                           Immunofluorescence- FN1, FOXF1, and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for FN1, FOXF1, and ACTA2.

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-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) 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, 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. 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 @ 4oC

 

Tissue Used:
LMH-15-D015-LLL-6C4.11
Age: 3 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 |2018-11-12T16:04:18+00:00March 15th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

8 Month-Old Human Lung Confocal Imaging Donor D086 FN1, FOXF1, and ACTA2

8 Month-Old Human Lung Confocal Imaging Donor D086 FN1, FOXF1, and ACTA2

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

Pryhuber_Lab

                                                Immunofluorescence- FN1, FOXF1, and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for FN1, FOXF1, and ACTA2.

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-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) 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, 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. 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 @ 4oC

 

Tissue Used:
LMH-15-D086-LLL-8B2.6
Age: 8 Months
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

By |2018-11-12T16:05:47+00:00March 15th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

13 Month-Old Human Lung Confocal Imaging Donor D083 FN1, FOXF1, and ACTA2

13 Month-Old Human Lung Confocal Imaging Donor D083 FN1, FOXF1, and ACTA2

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

Pryhuber_Lab

Immunofluorescence- FN1, FOXF1, and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for FN1, FOXF1, and ACTA2.

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-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) 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, 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. 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 @ 4oC

 

Tissue Used:
LMH-15-D083-LLL-10A3.6
Age: 13 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

By |2018-11-12T16:07:34+00:00March 15th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
Read More

3 Year-Old Human Lung Confocal Imaging Donor D053 FN1, FOXF1, and ACTA2

3 Year-Old Human Lung Confocal Imaging Donor D053 FN1, FOXF1, and ACTA2

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

Pryhuber_Lab

Immunofluorescence- FN1, FOXF1, and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for FN1, FOXF1, and ACTA2.

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-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) 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, 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. 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 @ 4oC

 

Tissue Used:
LMH-15-D053-LLL-5B2.28
Age: 3 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

By |2018-11-12T16:14:29+00:00March 15th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
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39 Month-Old Human Lung Confocal Imaging Donor D039 FN1, FOXF1, and ACTA2

39 Month-Old Human Lung Confocal Imaging Donor D039 FN1, FOXF1, and ACTA2

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

Pryhuber_Lab

                                                  Immunofluorescence- FN1, FOXF1, and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of human lung from HTC for FN1, FOXF1, and ACTA2.

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-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) 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, 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. 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 @ 4oC

 

 

Tissue Used:
LMH-15-D039-LLL-6B2.11
Age: 39 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

By |2018-11-12T16:13:10+00:00March 15th, 2018|Categories: BPD Confocal Imaging, Human Lung Disease Confocal Imaging, Postnatal Human Lung|Tags: , , |0 Comments
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Alveolar type II cells, also known as type II pneumocytes, are specialized epithelial cells found in the alveoli of the lungs, which are the tiny air sacs responsible for gas exchange. These cells play a crucial role in maintaining the structure and function of the respiratory system.

Alveolar type II cells are located within the alveolar walls of the lungs.

Alveolar type II cells are cuboidal or squamous in shape and are smaller than alveolar type I cells, which are responsible for gas exchange. They are characterized by the presence of microvilli on their apical surface, which increases their surface area for various functions.

One of the most important functions of alveolar type II cells is the production and secretion of pulmonary surfactant. Surfactant is a complex mixture of lipids and proteins that reduces the surface tension of the alveolar fluid, preventing the collapse of alveoli during exhalation. This property is crucial for maintaining the stability of the alveoli and preventing lung collapse, particularly at the end of expiration.

Alveolar type II cells serve as progenitor cells for alveolar type I cells. They have the ability to self-renew and differentiate into type I cells, helping in the repair and regeneration of the alveolar epithelium after injury or damage. This regenerative capacity is essential for maintaining the integrity of the respiratory barrier.

Alveolar type II cells also play a role in the innate immune response of the lungs. They can produce and secrete various immune molecules, including cytokines, chemokines, and antimicrobial peptides, in response to pathogens or inflammatory stimuli. These molecules help to recruit immune cells to the site of infection and promote the clearance of pathogens from the lungs.

Alveolar type II cells are involved in the regulation of ion and fluid balance within the alveoli. They express various ion channels and transporters that regulate the movement of ions, such as sodium and chloride, across the epithelial barrier. This process is important for maintaining the osmotic balance of the alveolar fluid and preventing the accumulation of fluid in the lungs (pulmonary edema).

The surfactant produced by alveolar type II cells consists mainly of phospholipids (such as dipalmitoylphosphatidylcholine, or DPPC) and surfactant proteins (SP-A, SP-B, SP-C, and SP-D). These components work together to reduce surface tension and maintain the stability of the alveoli.

The production and secretion of surfactant by alveolar type II cells are regulated by various factors, including mechanical stretch, glucocorticoid hormones, and certain signaling molecules (such as epinephrine and thyroid hormones). These regulatory mechanisms ensure that surfactant production is matched to the demands of respiration and the maintenance of lung function.