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1 Day-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

1 Day-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

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

Pryhuber_Lab

Immunofluorescence: SOX9, SOX2, and ACTA2 

 

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) 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-SOX9) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, 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:
LMH-D019-RLL-5B1.22_SOX9_SOX2_ACTA2
Gender: Male
Age: 1 Day                        

                       

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 |2018-10-02T11:47:41+00:00January 26th, 2018|Categories: 01 Day-Old, Homo sapiens Confocal Imaging|Tags: , , |0 Comments
Read More

20 Month-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

20 Month-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

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

Pryhuber_Lab

Immunofluorescence: SOX9, SOX2, and ACTA2 

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) 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-SOX9) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, 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:
LMH-16-D008-RLL-7B3.18
Gender: Female
Age: 20 Months                   

                       

 

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 |2018-10-03T14:51:31+00:00January 25th, 2018|Categories: 20 Month-Old, Homo sapiens Confocal Imaging|Tags: , , |0 Comments
Read More

1 Day-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

1 Day-Old Human Lung Confocal Imaging SOX9, SOX2 and ACTA2

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

Pryhuber_Lab

Immunofluorescence: SOX9, SOX2, and ACTA2 

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) 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-SOX9) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, 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:
LMH-16-D038-RLL-7A2.19
Gender: Female
Age: 1 Day                                  

                       

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 |2018-10-02T11:54:04+00:00January 25th, 2018|Categories: 01 Day-Old, Homo sapiens Confocal Imaging|Tags: , , |0 Comments
Read More

3 Year-Old Old Human Lung Confocal Imaging SOX9, SOX2 and TUBB3

3 Year-Old Old Human Lung Confocal Imaging SOX9, SOX2 and TUBB3

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

Pryhuber_Lab

                                            Immunofluorescence: SOX9, SOX2, and TUBB3

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and TUBB3 with antigen
retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz,
    Lot# C0317) dilute 1:100, for mouse anti-TUBB3 (anti-tubulin β 3, cat. # 801201, Biolegend) dilute 1:200 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-SOX9) at 1:200, Goat
    Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse
    IgG2A A21136, Lot# 1345055, TUBB3) 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:
LMH-16-D032-RLL-4A2.6
Gender: Female
Age: 3 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

 
 
 

Related Experiments

By |2018-09-26T17:23:36+00:00January 25th, 2018|Categories: 03 Year-Old, Homo sapiens Confocal Imaging|Tags: , , |0 Comments
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1 Day Human Lung SOX9, SOX2, and TUBB3 Confocal Imaging

1 Day Human Lung SOX9, SOX2, and TUBB3 Confocal Imaging

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

Pryhuber_Lab

Immunofluorescence: SOX9, SOX2, and TUBB3

Purpose:  Stain slides of 4µm frozen sections of human lung from HTC and from Pathology for SOX9, SOX2, and TUBB3 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  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-SOX9 (AB–5535, Lot # 2167153, Millipore) dilute 1:100, for mouse anti-SOX2 (SC-365823, Santa Cruz, Lot# C0317) dilute 1:100, for mouse anti-TUBB3 (anti-tubulin β 3, cat. # 801201, Biolegend) dilute 1:200 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-SOX9) at 1:200, Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-SOX2) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A A21136, Lot# 1345055, TUBB3) 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:   
LMH-16-D038-RLL-7A1.12
Gender: Female
Age: 1 Day

 

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 |2018-09-14T13:21:19+00:00January 24th, 2018|Categories: 01 Day-Old, Homo sapiens Confocal Imaging|Tags: , , |0 Comments
Read More

Postnatal Day 10 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 10 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- SOX9, NKX2.1 and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND10 lungs for SOX9, 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-SOX9 (AB-5535, Lot # 2167153, 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–SOX9) 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) 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.43

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

By |2018-08-14T19:14:50+00:00April 6th, 2017|Categories: Mus musculus, P10|Tags: , , , , |0 Comments
Read More

Postnatal Day 3 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 3 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- SOX9, NKX2.1 and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND03 lungs for SOX9, 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-SOX9 (AB-5535, Lot # 2167153, 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–SOX9) 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) 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.3.44

C57BL6 PND3

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

By |2018-08-14T19:12:03+00:00April 6th, 2017|Categories: Mus musculus, P03|Tags: , , , , |0 Comments
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Postnatal Day 10 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Single Cell RNA-Seq and Gene Info

Immunofluorescence- SOX9, NKX2.1 and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6  PND10 lungs for SOX9, 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-SOX9 (AB-5535, Lot # 2167153, 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–SOX9) 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) 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.16.92A.6L.34

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

By |2018-08-14T19:14:52+00:00March 22nd, 2017|Categories: Mus musculus, P10|Tags: , , , , |0 Comments
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Postnatal Day 10 C57BL6 SOX9, pHISTH3, and ACTA2 Confocal Imaging

Postnatal Day 10 C57BL6 SOX9, pHISTH3, and ACTA2 Confocal Imaging

Immunofluorescence SOX9, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND10 lungs for SOX9, pHISH3 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-SOX9 (AB-5535, Lot # 2167153, Millipore) dilute 1:100. For goat pHISH3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 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-SOX9), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-pHISH3) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1366514, 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 15 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 @ 4oC.

 

Tissue Used:   

LMM.16.84A.5L.19

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

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By |2018-08-14T19:14:54+00:00August 2nd, 2016|Categories: Mus musculus, P10|Tags: , , , , |0 Comments
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