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Postnatal Day 10 CALCA, SCGB1A1, and TUBA1A Imaging

Postnatal Day 10 CALCA, SCGB1A1, and TUBA1A Imaging

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

Pryhuber_Lab

 

Immunofluorescence CALCA, SCGB1A1, TUBA1A1

 

Purpose: Stain slides of 7µm frozen sections of PND10 C57Bl6 lungs for CALCA, SCGB1A1, TUBA1A1 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. Briefly equilibrate slides in Antigen Retrieval Buffer.
  2. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  3. Microwave according to instructions on microwave.
  4. Cool on countertop, 15 min.
  5. Rinse with dH2O
  6. 1X PBS, 5 min.
  7. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  8. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-CCSP (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A1(T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 4o

 

Tissue Used:
LMM.16.92A.6L.16
PND10 C57BL6            
Gender: Male
Weight= 4.1g

                                   

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 |2018-10-17T19:25:22+00:00August 1st, 2016|Categories: Mus musculus, P10|Tags: , , |0 Comments
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Postnatal Day 03 C57BL6 CALCA, SCGB1A1, and TUBA1A Confocal Imaging

Gene
Calca LungGens Ensemble Genecards NCBI The Human Protein Atlas
Scgb1a1a LungGens Ensemble
Genecards NCBI The Human Protein Atlas
Tuba1a 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 CALCA, SCGB1A1, TUBA1A1

 

Purpose: Stain slides of 7µm frozen sections of PND03 C57Bl6 lungs for CALCA, SCGB1A1, TUBA1A1 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. Briefly equilibrate slides in Antigen Retrieval Buffer.
  2. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  3. Microwave according to instructions on microwave.
  4. Cool on countertop, 15 min.
  5. Rinse with dH2O
  6. 1X PBS, 5 min.
  7. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  8. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-CCSP (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A1(T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 4o

 

Tissue Used:   

LMM.16.83A.4.19

PND03 C57BL6

Gender: Male

Weight= 2.1g

                                   

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 |2018-08-18T19:16:45+00:00August 1st, 2016|Categories: Mus musculus, P03|Tags: , , |0 Comments
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Postnatal Day 1 C57BL6 CALCA, SCGB1A1, and TUBA1A Confocal Imaging

Gene
Calca LungGens Ensemble Genecards NCBI The Human Protein Atlas
Scgb1a1a LungGens Ensemble
Genecards NCBI The Human Protein Atlas
Tuba1a LungGens Ensemble Genecards NCBI The Human Protein Atlas

 

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer.
  1. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  2. Microwave according to instructions on microwave.
  3. Cool on countertop, 15 min.
  4. Rinse with dH2O
  5. 1X PBS, 5 min.
  6. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  7. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti-TUBA1A (T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 4o

 

 

 

Tissue Used:    LMM.15.66A.6.73

PND01 C57BL6

Gender: Female

Weight= 1.14g

By |2018-08-17T23:13:42+00:00March 14th, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments
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Postnatal Day 28 C57BL6 SCGB1A1, CALCA, and TUBA1A Confocal Imaging

Gene
Calca LungGens Ensemble Genecards NCBI The Human Protein Atlas
Scgb1a1a LungGens Ensemble
Genecards NCBI The Human Protein Atlas
Tuba1a LungGens Ensemble Genecards NCBI The Human Protein Atlas

 

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. 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.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  4. Microwave according to instructions on microwave.
  5. Cool on countertop, 15 min.
  6. Rinse with dH2O.
  7. 1X PBS, 5 min.
  8. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  9. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-CCSP (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A1(T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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.

Tissue Used:   

LMM.15.16A.6.36

PND28

Gender: Female

Weight: 10.0g 

 

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 |2018-08-14T19:25:45+00:00April 12th, 2015|Categories: Mus musculus, P28|Tags: , , , , |0 Comments
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Postnatal Day 7 C57BL6 SCGB1A1, CALCA, and TUBA1A Confocal Imaging

Postnatal Day 7 C57BL6 SCGB1A1, CALCA, and TUBA1A Confocal Imaging

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin. 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.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  4. Microwave according to instructions on microwave.
  5. Cool on countertop, 15 min.
  6. Rinse with dH2O.
  7. 1X PBS, 5 min.
  8. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  9. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-CCSP (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A1(T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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.

 

Tissue Used:
LMM.15.12A.4.46
PND7 C57BL6
Gender: Male
Weight: 3.0g

 

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 |2018-12-06T13:32:19+00:00April 12th, 2015|Categories: Mus musculus, P07|Tags: , , , , , , , |0 Comments
Read More

E18.5 C57BL6 SCGB1A1, CALCA, and ACTA2 Confocal Imaging

E18.5 C57BL6 SCGB1A1, CALCA, and ACTA2 Confocal Imaging

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 –CALCA (CGRP) (C7113, Sigma) dilute 1:200 in blocking buffer, For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- ACTA2 (α Smooth muscle actin) (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin for 10 minutes in a µfuge, apply to tissue, 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 IgG1 (A21121, Lot# 1345050, for anti –CALCA (CGRP) ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, anti-SCGBA1A1) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2A (A21136, Lot# 1345055, ACTA2) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 a light sealed box  @ 4oC.

 

Tissue Used:

LMM.14.49.5.82 E18.5

Gender: Male

Crown-to-Rump Length: 22.0 mm

Weight=1.17g

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 |2018-08-17T22:44:06+00:00April 3rd, 2015|Categories: E18.5, Mus musculus|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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