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H&E Staining of 43 Year Old Human Lung D0037.05HP_2_24

Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Rinse slides 2X in dH2O for 20 dips each rinse.
  3. Rinsed slides for 3 minutes in running dH2O.
  4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  5. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  6. 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.
  7. Rinsed slides in dH2O 3X 20 dips.
  8. Rinsed slides in 70% ETOH for 20 dips.
  9. Rinsed slides in 80% ETOH for 20 dips.
  10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  11. 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.
  12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-2017-12-19_D0037.05HP_2_24

Gender: Male

Age: 43 Year Old

Race: Hispanic

Non-Smoker

By |2019-09-10T18:44:29+00:00February 27th, 2018|Categories: 47 Year Old, Adult Human Lung, H&E|Tags: , |0 Comments
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47 Year-Old Human Lung NKX2.1, AGER, and ACTA2 Confocal Imaging

Gene
Nkx2-1 LungGens Ensemble Genecards NCBI The Human Protein Atlas
AGER LungGens Ensemble Genecards NCBI The Human Protein Atlas
Acta2 LungGens Ensemble Genecards NCBI The Human Protein Atlas

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

Pryhuber_Lab

Immunofluorescence-  NKX2.1, AGER, and ACTA2

Purpose:  Stain slides of 7µm frozen sections of 47-year-old human lung, courtesy of Dr. Scott Randell, University of North Carolina, for NKX2.1, AGER, 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- NKX2.1  (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For rat anti-AGER (MAB 1179-500, RND Systems) 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-NKX2.1 Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-AGER) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, anti-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 proof box @ 4oC.

Tissue Used:    LMH-15-DD036-04HF-2.15

Gender: Male

Age: 47 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 |2018-02-26T17:16:54+00:00February 26th, 2018|Categories: 47 Year Old, Adult Human Lung|Tags: , , , , , |0 Comments
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H&E Staining 47 Year Old Human Lung DD036L-04HP-2

Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Rinse slides 2X in dH2O for 20 dips each rinse.
  3. Rinsed slides for 3 minutes in running dH2O.
  4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  5. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  6. 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.
  7. Rinsed slides in dH2O 3X 20 dips.
  8. Rinsed slides in 70% ETOH for 20 dips.
  9. Rinsed slides in 80% ETOH for 20 dips.
  10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  11. 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.
  12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-DD036L-04HP-2.18

Gender: Male

Age: 47 Year Old

Race: Caucasian

Non-Smoker

By |2019-09-10T18:50:41+00:00October 16th, 2015|Categories: 47 Year Old, Adult Human Lung, H&E|Tags: , |0 Comments
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H&E Staining of 47 Year Old Human Lung DD036L-04HP-5

Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Rinse slides 2X in dH2O for 20 dips each rinse.
  3. Rinsed slides for 3 minutes in running dH2O.
  4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  5. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  6. 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.
  7. Rinsed slides in dH2O 3X 20 dips.
  8. Rinsed slides in 70% ETOH for 20 dips.
  9. Rinsed slides in 80% ETOH for 20 dips.
  10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  11. 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.
  12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-DD036L-04HP-5.17

Gender: Male

Age: 47 Year Old

Race: Caucasian

Non-Smoker

By |2019-09-10T18:50:11+00:00October 16th, 2015|Categories: 47 Year Old, Adult Human Lung, H&E|Tags: , |0 Comments
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H&E Staining 47 Year Old Human Lung DD036L-04HF-2

Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Rinse slides 2X in dH2O for 20 dips each rinse.
  3. Rinsed slides for 3 minutes in running dH2O.
  4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  5. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  6. 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.
  7. Rinsed slides in dH2O 3X 20 dips.
  8. Rinsed slides in 70% ETOH for 20 dips.
  9. Rinsed slides in 80% ETOH for 20 dips.
  10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  11. 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.
  12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-DD036L-04HF-2.2

Gender: Male

Age: 47 Year Old

Race: Caucasian

Non-Smoker

By |2019-09-10T18:49:43+00:00October 7th, 2015|Categories: 47 Year Old, Adult Human Lung, H&E|Tags: , |0 Comments
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H&E Staining 47 Year Old Human Lung DD036L-04HF-1

Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Rinse slides 2X in dH2O for 20 dips each rinse.
  3. Rinsed slides for 3 minutes in running dH2O.
  4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
  5. Rinsed slides in tap water until water turned cleared then rinsed in dH2O
  6. 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.
  7. Rinsed slides in dH2O 3X 20 dips.
  8. Rinsed slides in 70% ETOH for 20 dips.
  9. Rinsed slides in 80% ETOH for 20 dips.
  10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
  11. 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.
  12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
  13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
  14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-DD036L-04HF-1-1, 17, 35

Gender: Male

Age: 47 Year Old

Race: Caucasian

Non-Smoker

By |2019-09-10T18:49:13+00:00July 20th, 2015|Categories: 47 Year Old, Adult Human Lung, H&E|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.

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