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Postnatal Day 3 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 3 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND03 lungs for CSPG4, EMCN, 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-CSPG4 (AB5320, Millipore) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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# 1387792, for anti-CSPG4) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-EMCN) 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 3X for 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:   

LMM.16.81A.3.26

PND03 C57BL6          

Gender: Male

Weight= 1.79g

 

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 |2018-08-14T19:12:05+00:00March 20th, 2017|Categories: Mus musculus, P03|Tags: , , , , , |0 Comments
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Postnatal Day 10 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 10 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND10 lungs for CSPG4, EMCN, 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-CSPG4 (AB5320, Millipore) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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# 1387792, for anti-CSPG4) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-EMCN) 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 3X for 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:   

LMM.16.84A.5L.20

PND10 C57BL6

Gender: Male

Weight= 4.5g

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 |2018-08-14T19:14:53+00:00March 20th, 2017|Categories: Mus musculus, P10|Tags: , , , , , |0 Comments
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Postnatal Day 14 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 14 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND14 lungs for CSPG4, EMCN, 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-CSPG4 (AB5320, Millipore) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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-CSPG4) at 1:200, Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti- EMCN) 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 3X for 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).
  1. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  2. Store slides in light sealed box @ 4oC

 

 Tissue Used:  LMM.15.34A.5L.55

C57BL6 PND14

Gender: Male

Weight: 5.91g

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 |2018-08-19T23:19:24+00:00April 6th, 2016|Categories: Mus musculus, P14|Tags: , , , , , , , , |0 Comments
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Postnatal Day 14 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 14 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, NKX2.1, and ACTA2

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND14 lungs for CSPG4, NKX2.1, 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% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-CSPG4 (AB5320, Millipore) dilute 1:100. For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, 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-CSPG4), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:500, 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 3X for 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.15.33A.6L.52

C57BL6 PND14

Gender: Female

Weight: 6.75g

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 |2018-08-19T23:20:04+00:00April 4th, 2016|Categories: Mus musculus, P14|Tags: , , , , , , |0 Comments
Read More

Postnatal Day 1 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for CSPG4, EMCN, 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-CSPG4 (R1231) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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-CSPG4) at 1:200, Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti- EMCN) 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 3X for 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.15.66A.6.55

PND01 C57BL6

Gender: Female

Weight= 1.14g

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 |2018-08-17T23:27:50+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , , , |0 Comments
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Postnatal Day 1 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, NKX2.1, and ACTA2

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for CSPG4, NKX2.1, 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% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-CSPG4 (AB5320, Millipore) dilute 1:100. For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, 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-CSPG4) 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:500, 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 3X for 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:   LMM.15.66A.6.56

PND01 C57BL6

Gender: Female

Weight= 1.14g

 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 |2018-08-17T23:16:41+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments
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Postnatal Day 28 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 28 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND28 lungs for CSPG4, EMCN, 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-CSPG4 (R1231) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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-CSPG4) at 1:200, Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti- EMCN) 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 3X for 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.15.20A.1.31

PND28 C57BL6

Gender: Female

 Weight= 10.2g

 

 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 6.0J

Related Experiments

By |2018-08-14T19:25:46+00:00April 12th, 2015|Categories: Mus musculus, P28|Tags: , , , , , |0 Comments
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Postnatal Day 7 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 7 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND07 lungs for CSPG4, EMCN, 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-CSPG4 (R1231) dilute 1:100. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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-CSPG4) at 1:200, Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti- EMCN) 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 3X for 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.15.23A.4L.45

PND7 C57BL6

 Gender: Male

Weight: 3.3g

 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 |2018-08-18T19:56:44+00:00April 12th, 2015|Categories: Mus musculus, P07|Tags: , , , , , |0 Comments
Read More

Postnatal Day 7 C57Bl6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 7 C57Bl6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, NKX2.1, and ACTA2

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND07 lungs for CSPG4, NKX2.1, 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% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-CSPG4 (AB5320, Millipore) dilute 1:100. For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, 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-CSPG4), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:500, 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 3X for 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.15.17A.5.24

                            PND7 C57BL6

                            Gender: Male

                            Weight= 4.00g 

 

 

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

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By |2018-08-18T19:57:34+00:00April 12th, 2015|Categories: Mus musculus, P07|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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