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Postnatal Day 14 C57BL6 SFTPC, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 14 C57BL6 SFTPC, pHIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence SFTPC, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND14 lungs for SFTPC, 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- SFTPC  (RB458, BL 8/24/11) 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-SFTPC), 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 a light sealed box @ 4oC.

 

Tissue Used:    LMM.15.33A.6L.48

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 |2018-08-14T19:23:38+00:00April 4th, 2016|Categories: Mus musculus, P14|Tags: , , , , , , |0 Comments
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Postnatal Day 14 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 14 C57BL6 HOPX, pHIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence HOPX, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND14 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 a light sealed box @ 4oC.

 

Tissue Used:    LMM.15.34A.5L.51

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 |2018-08-14T19:23:39+00:00April 4th, 2016|Categories: Mus musculus, P14|Tags: , , , , , |0 Comments
Read More

Postnatal Day 14 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 14 C57BL6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence- NKX2.1, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND14 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), 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 a light sealed box @ 4oC.

Tissue Used:    LMM.15.34A.5L.45

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 |2018-08-19T23:26:39+00:00April 4th, 2016|Categories: Mus musculus, P14|Tags: , , , , , , |0 Comments
Read More

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

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

Immunofluorescence SFTPC, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND01 lungs for SFTPC, 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-SFTPC (RB458, BL 8/24/11, in house) 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- SFTPC) 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. 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.15.69A.5.38

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 |2018-08-17T23:12:38+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments
Read More

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

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

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

By |2018-08-17T23:11:48+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , |0 Comments
Read More

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

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

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

By |2018-08-17T23:23:22+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: , , , , , , |0 Comments
Read More

E18.5 HOPX, phospho-HIST3H3, and ACTA2 Confocal Imaging

Single Cell RNA-Seq and Gene Info

Immunofluorescence HOPX, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57Bl/6 E18.5 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 for 15 min at 96oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  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 Hopx (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200. For goat p-Histone H3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin for 5 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 , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for Hopx), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, p-Histone H3) 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.
  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.

Tissue Used:

LMM.14.38.2.44 E18.5

Gender: Male

Crown-to-Rump Length: 21.0 mm

Weight=1.06g

 

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

By |2018-08-20T23:42:20+00:00May 22nd, 2015|Categories: E18.5, Mus musculus|Tags: , , , , |0 Comments
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Postnatal Day 28 C57Bl6 SFTPC, phospho-Hist3H3, and ACTA2 Confocal Imaging

Postnatal Day 28 C57Bl6 SFTPC, phospho-Hist3H3, and ACTA2 Confocal Imaging

Immunofluorescence SFTPC, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND28 lungs for SFTPC, 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-SFTPC (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-SFTPC), 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 a light sealed box @ 4oC.

 

Tissue Used:  

LMM.15.20A.1.29

C57BL6 PND28

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 6.0

 

Related Experiments

By |2018-08-14T19:25:43+00:00April 19th, 2015|Categories: Mus musculus, P28|Tags: , , , , , |0 Comments
Read More

Postnatal Day 28 C57Bl6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Postnatal Day 28 C57Bl6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence- NKX2.1, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND28 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 (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), 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 a light sealed box  @ 4oC. 

 

Tissue Used:  

LMM.15.20A.1.27

C57BL6 PND28

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 6.0

 

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By |2018-08-14T19:25:44+00:00April 19th, 2015|Categories: Mus musculus, P28|Tags: , , , , , , |0 Comments
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