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Confocal Imaging for C57BL6

PND01 NKX2.1, ABCA3, and ACTA2 Confocal Imaging

Gene
Nkx2.1 LungGens Ensemble Genecards NCBI The Human Protein Atlas
FOXF1 LungGens Ensemble Genecards NCBI The Human Protein Atlas
Acta2 LungGens Ensemble Genecards NCBI The Human Protein Atlas

Immunofluorescence- NKX2.1, ABCA3, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND01 lungs for NKX2.1, ABCA3, 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-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, for guinea pig anti-ABCA3 (G985, BL 3/10/05, Seven Hills) 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti- NKX2.1) at 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-ABCA3) 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.
  1. Rinse in PBS-T 3X, 5 min.
  2. Dilute DAPI 1:2000 and apply to slides for 15 min.
  3. Wash in PBS-T 3X, 5 min.
  4. Rinse slides in 0.1M PB, 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 light sealed box @ 4oC

Tissue Used:   

LMM.15.66A.6.50

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

By |2018-08-14T19:09:28+00:00March 23rd, 2018|Categories: Mus musculus, P01|Tags: , , |0 Comments
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E16.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

E16.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

 

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

Purpose:  Stain slides of 7µm frozen sections of C57Bl/6J E16.5 mouse lung for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC
 

Related Experiments

By |2018-08-16T02:46:33+00:00February 23rd, 2018|Categories: E16.5, Mus musculus|Tags: , , |0 Comments
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E18.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

E18.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57Bl/6J mouse lung E18.5 for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.14.38.2.29
E18.5 C57BL6
Crown-to-rump: 21mm
Gender: Male
Weight: 61.06g

 

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 |2018-08-14T19:02:59+00:00February 23rd, 2018|Categories: E18.5, Mus musculus|Tags: , , |0 Comments
Read More

PND01 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND01 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND01 mouse lung  for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.15.69A.5.23

PND01 C57BL6          

Gender: Male

Weight= 1.36g

 

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 |2018-08-14T19:09:28+00:00February 23rd, 2018|Categories: Mus musculus, P01|Tags: , , |0 Comments
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Postnatal Day 28 Mouse Lung Second Harmonic Imaging

Postnatal Day 28 mouse lungs were inflation fixed with 4% PFA and incubated overnight at 4oC. After overnight fixation, lungs were washed several times DEPC-treated 1X PBS and dehydrated through a Methanol (MeOH) series to 100% MeOH. Lung tissue was then washed in 3 changes of 100% MeOH for 30 minutes. Once tissue is equilibrated in 100% MeOH, remove air bubbles by squeezing out the air bubbles or by using a vacuum.

Clear in BA:BB 1:2  (1 part benzyl alcohol: 2 parts benzyl benzoate). For a 1.5cm x 1.5 cm x 5mm piece of tissue, it takes ~ 1 hour to clear.

Transfer lungs to fresh BA:BB. Image on a multiphoton microscope with glycerol immersion objectives. Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample. We use either a 10X NA 0.5 WD 5.5mm or a 20X NA 1.0 WD 8.2mm.  Image using backscattered SHG with GaAsP NDDs using 840nm excitation and 420/20 filter for SHG, 525/50 for autofluorescence and a 585/50 for pigment (pigment, carbon particles and other debris will “explode” under IR excitation and needs to be captured to remove from final datasets). Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample.

By |2018-08-14T19:25:38+00:00February 23rd, 2018|Categories: Mus musculus, P28, Uncategorized|0 Comments
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PND10 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND10 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND10 mouse lung  for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.16.92A.6L.57

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 |2018-08-14T19:14:59+00:00February 23rd, 2018|Categories: Mus musculus, P10|Tags: , , |0 Comments
Read More

PND03 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND03 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND03 mouse lung for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.16.83A.4.58

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

Related Experiments

By |2018-07-02T13:29:03+00:00February 23rd, 2018|Categories: P03|Tags: , , |0 Comments
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PND14 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND14 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND14 mouse lung for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.15.34A.5L.65

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 |2018-07-02T13:38:26+00:00February 22nd, 2018|Categories: P14|Tags: , , |0 Comments
Read More

PND28 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND28 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND28 mouse lung for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.15.24A.6L.75

PND28 C57BL6

Gender: Male

Weight: 10.5g

 

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 |2018-08-14T19:25:48+00:00February 22nd, 2018|Categories: Mus musculus, P28|Tags: , |0 Comments
Read More

PND7 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND7 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6 PND07 mouse lung for NKX2.1, FOXF1 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 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 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-FOXF1), 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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.15.27A.7L.59

PND07  C57BL6

Gender: Male

Weight: 4.4g

 

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 |2018-07-02T13:32:15+00:00February 22nd, 2018|Categories: 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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