Alveolar Gas ExchangeArtboard 2@2x

Menu

Postnatal Day 1 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence-LYVE1, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for LYVE1, EMCN, and ACTA2

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-LYVE1 (AB14917, ABCAM) 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-LYVE1), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-EMCN) 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. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  4. Rinse in PBS-T 3X, 5 min.
  5. Dilute DAPI 1:2000 and apply to slides for 15 min.
  6. Wash in PBS-T 3X, 5 min.
  7. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  10. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  11. Store slides in light sealed box @ 4oC.

Tissue Used:    LMM.15.66A.6.60

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

  • E16.5 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
    E16.5 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
    E18.5 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 LYVE1_EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 LYVE1_EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 LYVE1, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 LYVE1_EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 LYVE1_EMCN, and ACTA2 Confocal Imaging
By |2018-08-17T23:17:45+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments

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

The requested content cannot be found

Immunofluorescence-HOPX, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57Bl/6 PND01 lungs for HOPX, EMCN, and ACTA2

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-HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, 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-HOPX), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-EMCN) 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. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  4. Rinse in PBS-T 3X, 5 min.
  5. Dilute DAPI 1:2000 and apply to slides for 15 min.
  6. Wash in PBS-T 3X, 5 min.
  7. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  10. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  11. Store slides in light sealed box at 4o

Tissue Used:    LMM.15.69A.5.32

PND01 C57BL6           

Gender: Male

Weight= 1.36g

By |2018-08-17T23:26:12+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments

Postnatal Day 01 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 01 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence HOPX, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND01 lungs for HOPX, 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-HOPX (1231, Lot# 0912A) dilute 1:200. 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-HOPX), 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. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  4. Rinse in PBS-T 3X, 5 min.
  5. Dilute DAPI 1:2000 and apply to slides for 15 min.
  6. Wash in PBS-T 3X, 5 min.
  7. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  10. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  11. Store slides in light sealed box @ 4oC.

 

Tissue Used:    LMM.15.66A.6.58

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

  • E16.5 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
    E16.5 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
    E18.5 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging
  • Postnatal Day 03 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 03 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 HOPX, pHISH3, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 HOPX, pHISTH3, and ACTA2 Confocal Imaging
By |2018-08-17T23:11:48+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , |0 Comments

Postnatal Day 1 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- NKX2.1, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND01 lungs for NKX2.1, 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- NKX2.1  (RB TTF-1 1231, Lot# 0912A) dilute 1:1000. 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-1) dilute 1:200, 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. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  4. Rinse in PBS-T 3X, 5 min.
  5. Dilute DAPI 1:2000 and apply to slides for 15 min.
  6. Wash in PBS-T 3X, 5 min.
  7. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  10. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  11. Store slides in light sealed box @ 4oC.

Tissue Used:    LMM.15.69A.5.31

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

  • E16.5 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
    E16.5 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
    E18.5 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging
  • Postnatal Day 03 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 03 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 NKX2.1, pHISH3, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 NKX2.1, pHISTH3, and ACTA2 Confocal Imaging
By |2018-08-17T23:23:22+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments

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

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

The requested content cannot be found

Immunofluorescence- CSPG4EMCNand ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for CSPG4EMCNand 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

  • E16.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    E16.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    E18.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 3 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 3 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging
By |2018-08-17T23:27:50+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , , , |0 Comments

Postnatal Day 1 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- LYVE1, NKX2.1, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for LYVE1, 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-LYVE1 (AB14917, ABCAM) dilute 1:100. For guinea pig anti-NKX2.1 (G237, Bl. 8/25/11) 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 Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-LYVE1) at 1:200, Donkey 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.
  1. Store slides in light sealed box @ 4oC

 

 Tissue Used:    LMM.15.66A.6.76

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

  • E16.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    E16.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    E18.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 03 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 03 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 ARL13B, EMCN, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 ARL13B, EMCN, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging
By |2018-08-14T19:09:31+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , |0 Comments

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

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

The requested content cannot be found

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

  • E16.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    E16.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    E18.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 03 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 03 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 7 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 7 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
  • Postnatal Day 28 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
    Postnatal Day 28 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging
By |2018-08-17T23:16:41+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments

Postnatal Day 1 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging

Postnatal Day 1 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence SFTPC, SCGB1A1, and ACTA2

 Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND01 lung for SFTPC, SCGB1A1, 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 for 5 min.
  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-SFTPC (RB458, BL 8/24/11, in house) dilute 1:1000, 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 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-SFTPC), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-SCGB1A1) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1549801, ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubate 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.5.44

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

  • E16.5 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
    E16.5 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
  • E18.5 C57BL6 SCGB1A1,SFTPC, and ACTA2 Confocal Imaging
    E18.5 C57BL6 SCGB1A1,SFTPC, and ACTA2 Confocal Imaging
  • Postnatal Day 1 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
    Postnatal Day 1 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
  • Postnatal Day 03 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
    Postnatal Day 03 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
  • Post Natal Day 7 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
    Post Natal Day 7 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
  • Postnatal Day 10 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
    Postnatal Day 10 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
  • Postnatal Day 14 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
    Postnatal Day 14 C57BL6 SFTPC, SCGB1A1, and ACTA2 Confocal Imaging
  • Post Natal Day 28 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
    Post Natal Day 28 C57BL6 SCGB1A1, SFTPC, and ACTA2 Confocal Imaging
By |2018-08-17T23:20:24+00:00March 14th, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments

Postnatal Day 1 C57BL6 CALCA, SCGB1A1, and TUBA1A Confocal Imaging

Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
20x Tile Scan
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
20x
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
60x
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
20x
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
60x
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
20x
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
Post Natal Day 1 Confocal Imaging Showing Protein Expression of Calca, Scgb1a1, and Tuba1a Genes
60x
The requested content cannot be found

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

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

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

The requested content cannot be found

Immunofluorescence- HOPX, NKX2.1, and ACTA2

 

Purpose:  Stain slides of frozen sections of C57BL6 PND01 lung for HOPX, 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- HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, For guinea pig anti- anti-NKX2.1 (G237, Bl. 8/25/11) 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480,  for anti- HOPX), 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, 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. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  2. Rinse in PBS-T 3X, 5 min.
  3. Dilute DAPI 1:2000 and apply to slides for 15 min.
  4. Wash in PBS-T 3X, 5 min.
  5. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  6. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  7. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  8. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  9. Store slides in light sealed box at 4oC.

Tissue Used:M.15.69A.5.34

PND01 C57BL6

Gender: Male

Weight= 1.36g

By |2018-08-17T23:27:08+00:00March 14th, 2016|Categories: Mus musculus, P01|Tags: , , , , , |0 Comments