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4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging

4 Year-Old Human Lung KRT14, MUC5B, and TUBA4A Confocal Imaging

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

Pryhuber_Lab

Immunofluorescence-  KRT14, MUC5B, and TUBA4A

 

Purpose:  Stain slides of 7µm frozen slides of human lung from the HTC for KRT14, MUC5B, and TUBA4A with antigen retrieval.

Day 1

  1. Frozen slides are rehydrated 2X in 1X PBS for 5 minutes each, followed by 5 minutes in 4% paraformaldehyde, followed by a wash in 1X PBS for 5 minutes.
  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 rabbit anti-MUC5B (SC-20119, Lot # B1003 Santa Cruz) Dilute 1:100, For mouse anti-TUBA4A (Acetylated Tubulin, T7451, Sigma) dilute 1:1000 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, Goat Alexa Fluor 488 anti-mouse IgG3 (A21151, Lot# 1563682,  for anti-KRT14) at 1:200, Goat Alexa Fluor 555 anti-rabbit IgG (A21428, Lot# 1608466 for anti-MUC5B) at 1:200, Goat Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA4A)  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.
  10. Store slides at 4oC in a light sealed box.

 

Tissue Used:
LMH-16-D007-LLL-5C4.11
Gender: Male
Age: 4 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

By |2020-05-21T18:15:13+00:00May 21st, 2020|Categories: Uncategorized|0 Comments
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Alcian Blue Staining from PBR 3653 Patient with Cystic Fibrosis

Alcian Blue Staining from PBR 3653 Patient with Cystic Fibrosis

 

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

Pryhuber_Lab

 

  1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
  2. Rinsed slides for 3 minutes in running dH2O.
  3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
  4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
  5. Wash slides under cold running tap water for 10 minutes.
  6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
  7. Wash under flowing dH2O until excess Nuclear Fast Red is washed away.
  8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
  9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
  10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
  11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:
Alcian Blue Staining from PBR 3653 Patient with Cystic Fibrosis

By |2019-11-13T16:34:11+00:00November 13th, 2019|Categories: Uncategorized|0 Comments
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H&E Staining from PBR 3383 Patient with Cystic Fibrosis

H&E Staining from PBR 3383 Patient with Cystic Fibrosis

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

Pryhuber_Lab

  1. Paraffin sections were placed at 60oC overnight to melt paraffin.
  2. Paraffin sections were then placed in xylene (Xylenes Reagent Grade ACS Histology/Lab Grade Cat # 399000000 Pharma-AAPER) 3X for 10 minutes each.
  3. Rinsed slides in a series of 100% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  4. Rinsed slides in a series of 95% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  5. Rinsed slides in 70% ETOH for 3 min.
  6. Rinsed slides for 3 minutes in running dH2O.
  7. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  8. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  9. Rinsed slides in Lithium Carbonate: 20 dips. (Lithium Carbonate ACS Grade; Cat # 255823-500G Sigma Aldrich). Lithium carbonate is prepared by over-saturating in dH2O.
  10. Rinsed slides in dH2O 3X 20 dips.
  11. Rinsed slides in 70% ETOH for 20 dips.
  12. Rinsed slides in 80% ETOH for 20 dips.
  13. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  14. Rinsed slides in a series of 95% ETOH washes. 10 dips for the first and second washes, 15 dips in the third and fourth washes.
  15. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  16. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  17. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

 

Tissue Used:
PBR 3383 Patient with Cystic Fibrosis

By |2019-11-08T20:02:41+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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H&E Staining from PBR 3653 Patient with Cystic Fibrosis

H&E Staining from PBR 3653 Patient with Cystic Fibrosis

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

Pryhuber_Lab

  1. Paraffin sections were placed at 60oC overnight to melt paraffin.
  2. Paraffin sections were then placed in xylene (Xylenes Reagent Grade ACS Histology/Lab Grade Cat # 399000000 Pharma-AAPER) 3X for 10 minutes each.
  3. Rinsed slides in a series of 100% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  4. Rinsed slides in a series of 95% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  5. Rinsed slides in 70% ETOH for 3 min.
  6. Rinsed slides for 3 minutes in running dH2O.
  7. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  8. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  9. Rinsed slides in Lithium Carbonate: 20 dips. (Lithium Carbonate ACS Grade; Cat # 255823-500G Sigma Aldrich). Lithium carbonate is prepared by over-saturating in dH2O.
  10. Rinsed slides in dH2O 3X 20 dips.
  11. Rinsed slides in 70% ETOH for 20 dips.
  12. Rinsed slides in 80% ETOH for 20 dips.
  13. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  14. Rinsed slides in a series of 95% ETOH washes. 10 dips for the first and second washes, 15 dips in the third and fourth washes.
  15. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  16. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  17. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

 

Tissue Used:
PBR 3653 Patient with Cystic Fibrosis

By |2019-11-08T20:00:39+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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H&E Staining of 31 Year-Old Human Donor 10

H&E Staining of 31 Year-Old Human Donor 10

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

Pryhuber_Lab

  1. Paraffin sections were placed at 60oC overnight to melt paraffin.
  2. Paraffin sections were then placed in xylene (Xylenes Reagent Grade ACS Histology/Lab Grade Cat # 399000000 Pharma-AAPER) 3X for 10 minutes each.
  3. Rinsed slides in a series of 100% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  4. Rinsed slides in a series of 95% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  5. Rinsed slides in 70% ETOH for 3 min.
  6. Rinsed slides for 3 minutes in running dH2O.
  7. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  8. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  9. Rinsed slides in Lithium Carbonate: 20 dips. (Lithium Carbonate ACS Grade; Cat # 255823-500G Sigma Aldrich). Lithium carbonate is prepared by over-saturating in dH2O.
  10. Rinsed slides in dH2O 3X 20 dips.
  11. Rinsed slides in 70% ETOH for 20 dips.
  12. Rinsed slides in 80% ETOH for 20 dips.
  13. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  14. Rinsed slides in a series of 95% ETOH washes. 10 dips for the first and second washes, 15 dips in the third and fourth washes.
  15. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  16. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  17. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

 

Tissue Used:
LMH-18-D010
Age: 31 Years

By |2019-11-08T19:57:50+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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H&E Staining of 27 Year-Old Human Donor 3

H&E Staining of 27 Year-Old Human Donor 3

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

Pryhuber_Lab

  1. Paraffin sections were placed at 60oC overnight to melt paraffin.
  2. Paraffin sections were then placed in xylene (Xylenes Reagent Grade ACS Histology/Lab Grade Cat # 399000000 Pharma-AAPER) 3X for 10 minutes each.
  3. Rinsed slides in a series of 100% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  4. Rinsed slides in a series of 95% ETOH washes. 20 dips for the first wash, 3 minutes for the second wash, and 20 dips for the third wash.
  5. Rinsed slides in 70% ETOH for 3 min.
  6. Rinsed slides for 3 minutes in running dH2O.
  7. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  8. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  9. Rinsed slides in Lithium Carbonate: 20 dips. (Lithium Carbonate ACS Grade; Cat # 255823-500G Sigma Aldrich). Lithium carbonate is prepared by over-saturating in dH2O.
  10. Rinsed slides in dH2O 3X 20 dips.
  11. Rinsed slides in 70% ETOH for 20 dips.
  12. Rinsed slides in 80% ETOH for 20 dips.
  13. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  14. Rinsed slides in a series of 95% ETOH washes. 10 dips for the first and second washes, 15 dips in the third and fourth washes.
  15. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  16. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  17. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

 

Tissue Used:
LMH-18-D003
Age: 27 Years

By |2019-11-08T19:55:44+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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Alcian Blue Staining of 27 Year-Old Human Donor 3

Alcian Blue Staining of 27 Year-Old Human Donor 3

 

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

Pryhuber_Lab

 

  1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
  2. Rinsed slides for 3 minutes in running dH2O.
  3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
  4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
  5. Wash slides under cold running tap water for 10 minutes.
  6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
  7. Wash under flowing dH2O until excess Nuclear Fast Red is washed away.
  8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
  9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
  10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
  11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:
LMH-18-D003
Age: 27 Years

By |2019-11-08T19:51:36+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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Alcian Blue Staining of 31 Year-Old Human Donor 10

Alcian Blue Staining of 31 Year-Old Human Donor 10

 

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

Pryhuber_Lab

 

  1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
  2. Rinsed slides for 3 minutes in running dH2O.
  3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
  4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
  5. Wash slides under cold running tap water for 10 minutes.
  6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
  7. Wash under flowing dH2O until excess Nuclear Fast Red is washed away.
  8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
  9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
  10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
  11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:
LMH-18-D010
Age: 31 Years

By |2019-11-08T19:49:11+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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Alcian Blue Staining from PBR 3383 Patient with Cystic Fibrosis

Alcian Blue Staining from PBR 3383 Patient with Cystic Fibrosis

 

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

Pryhuber_Lab

 

  1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
  2. Rinsed slides for 3 minutes in running dH2O.
  3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
  4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
  5. Wash slides under cold running tap water for 10 minutes.
  6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
  7. Wash under flowing dH2O until excess Nuclear Fast Red is washed away.
  8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
  9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
  10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
  11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:
PBR 3383 Patient with Cystic Fibrosis

By |2019-11-08T19:44:59+00:00November 8th, 2019|Categories: Uncategorized|0 Comments
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Alcian Blue Staining of 29 Year-Old Human Donor D175-10C1.21

Alcian Blue Staining of 29 Year-Old Human Donor D175-10C1.21

 

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

Pryhuber_Lab

 

  1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
  2. Rinsed slides for 3 minutes in running dH2O.
  3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
  4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
  5. Wash slides under cold running tap water for 10 minutes.
  6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
  7. Wash under flowing dH2O until excess Nuclear Fast Red is washed away.
  8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
  9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
  10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
  11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:
LMH-18-D175-RLL-10C1.21
Gender: Female
Age: 29 Years

By |2019-11-06T19:13:15+00:00November 6th, 2019|Categories: Uncategorized|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.

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