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E16.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging

E16.5 C57BL6 LYVE1, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- LYVE1, NKX2.1, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J E16.5 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. 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-LYVE1), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:500, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, anti-ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Rinse in PBS-T 3X, 5 min.
  4. Dilute DAPI 1:2000 and apply to slides for 10 min.
  5. Wash in PBS-T 3X, 5 min.
  6. Rinse slides in 0.1M PB 3X for 5 min.
  7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  9. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  10. Store slides in a light sealed box @ 4oC.

 

 Tissue Used:    LMM.14.24.4.24

                             E16.5 C57BL6

                            Gender: Male

                            Crown-to-Rump= 16.0mm

                             Weight= 0.592g

 

 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 |2018-08-17T22:07:57+00:00April 15th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , , |0 Comments
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E16.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

E16.5 C57BL6 CSPG4, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, NKX2.1, and ACTA2

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J E16.5 lungs for CSPG4, NKX2.1, and ACTA2 with antigen retrieval.

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96oC. *We usually do a series of three runs (6-7 minutes each run) to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  5. Microwave according to instructions on microwave.
  6. Cool on countertop, 15 min.
  7. Rinse with dH2O
  8. 1X PBS, 5 min.
  9. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-CSPG4 (AB5320, Millipore) dilute 1:100. For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-CSPG4), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:500, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1549801, anti-ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Rinse in PBS-T 3X, 5 min.
  4. Dilute DAPI 1:2000 and apply to slides for 10 min.
  5. Wash in PBS-T 3X, 5 min.
  6. Rinse slides in 0.1M PB 3X for 5 min.
  7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  9. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  10. Store slides in a light sealed box @ 4oC. 

 

 Tissue Used:    LMM.14.24.4.41

E16.5 C57BL6

                        Gender: Male

                        Crown-to-rump: 16.0 mm

                        Weight= 0.592g

 

 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 |2018-08-17T22:03:07+00:00April 14th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , , |0 Comments
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E16.5 C57BL6 HOPX, EMCN, and ACTA2 Confocal Imaging

E16.5 C57BL6 HOPX, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence-HOPX, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J E16.5 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# 1608521, for anti-HOPX), 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, 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, 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.14.24.4.48

E16.5 C57BL6

Gender: Male

Crown-to-rump: 16.0mm

Weight: 0.592

 

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 |2018-08-17T22:00:37+00:00April 14th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , , |0 Comments
Read More

E16.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

E16.5 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

Immunofluorescence- CSPG4, EMCN, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J E16.5 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96oC. *We usually do a series of three runs (6-7 minutes each run) to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  5. Microwave according to instructions on microwave.
  6. Cool on countertop, 15 min.
  7. Rinse with dH2O
  8. 1X PBS, 5 min.
  9. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-CSPG4 (AB5320, milipore, anti-NG2) 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.14.24.4.48

E16.5 C57BL6

Gender: Male

Crown-to-rump: 16.0mm

 Weight= 0.592g

  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 |2018-08-17T22:11:46+00:00April 14th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , , , |0 Comments
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E16.5 C57BL6 ARL13B, NKX2.1, and ACTA2 Confocal Imaging

E16.5 C57BL6 ARL13B, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence-ARL13B, NKX2.1, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 E16.5 lungs for ARL13B, 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.Briefly equilibrate slides in Antigen Retrieval Buffer.
  2. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  3. 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)
  4. Microwave according to instructions on microwave.
  5. Cool on countertop, 15 min.
  6. Rinse with dH2O
  7. 1X PBS, 5 min.
  8. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  9. For Rabbit anti-ARL13B (17711-AP) dilute 1:100, For guinea pig anti-NKX2.1(G237, Bl. 10/7/11) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 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-rabbit IgG (A11034, Lot# 1298480, for anti- ARL13B), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, anti-NKX2.1) at 1:200 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 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 in a light sealed box @ 4oC.

Tissue Used:   

LMM.14.36.1.63 E16.5

Gender: Female

Crown-to-Rump Length: 15.0 mm

Weight=0.540g

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 |2018-08-17T21:47:21+00:00March 6th, 2015|Categories: E16.5, Mus musculus|0 Comments
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E16.5 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging

E16.5 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging

 
 

Immunofluorescence- HOPX, SFTPC, and ACTA2

 

Purpose: Stain slides of 5µm paraffin sections of C57BL/6J E16.5 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval.

Day 1

 
  1. Paraffin sections are placed at 60 oC for 2 hours to overnight to melt paraffin. 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.
  2. For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96 oC. *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 dH 2O
  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-SFTPC (SC-7706, Santa Cruz) dilute 1:100, 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 @ 4o C.
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- SFTPC) 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 a light sealed box @ 4o C.
 
Tissue Used:  

LMM.14.57.4.39 E16.5

Gender: Female

Crown-to-Rump Length: 16.0 mm

Weight=0.574g

 
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 dH2 O
pH 6.0
 
By |2018-08-17T22:05:19+00:00March 6th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , |0 Comments
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E16.5 C57Bl6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

E16.5 C57Bl6 NKX2.1, pHIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence- NKX2.1, pHISH3 and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL/6J E16.5 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 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 anti-NKX2.1 (1231, Lot# 0912A) dilute 1:1000. For goat anti-p-Hist3H3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2  (ACTA2) (A5228, Sigma) dilute 1:2000 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 , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1387792, for anti-NKX2.1), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, for anti- p-HIST3H3) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1366514, for 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 a light sealed box @ 4oC.

Tissue Used:  

LMM.14.24.4.30 E16.5

Gender: Male

Crown-to-Rump Length: 16.0 mm

Weight=0.592g

 

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 |2018-08-14T16:50:12+00:00February 18th, 2015|Categories: E16.5, Mus musculus|0 Comments
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E16.5 C57Bl6 SFTPC, phospho-Hist3H3, and ACTA2 Confocal Imaging

E16.5 C57Bl6 SFTPC, phospho-Hist3H3, and ACTA2 Confocal Imaging

Immunofluorescence SFTPC, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J E16.5 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 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 anti-SFTPC (RB458, BL 8/24/11, in house) dilute 1:1000. For goat anti-p-Hist3H3 (SC-12927, Lot # L2013, Santa Cruz) dilute 1:500, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 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 , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, for anti-SFTPC), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-p-Hist3H3) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1549801, for 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 a light sealed box  @ 4oC.

 

 

Tissue Used:    Appendix

LMM.14.24.4.49 E16.5

Gender: Male

Crown-to-Rump Length: 16.0 mm

Weight=0.592g

 

 

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 |2018-08-14T16:50:12+00:00February 18th, 2015|Categories: E16.5, Mus musculus|0 Comments
Read More

E16.5 HOPX, phospho-HIST3H3, and ACTA2 Confocal Imaging

E16.5 HOPX, phospho-HIST3H3, and ACTA2 Confocal Imaging

Immunofluorescence HOPX, pHISH3 and ACTA2

 

Purpose: Stain slides of 7µm frozen sections of C57BL/6J E16.5 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.
  10. Store slides in a light sealed box @ 4oC.

 

Tissue Used:   

LMM.14.24.4.40 E16.5

Gender: Male

Crown-to-Rump Length: 16.0 mm

Weight=0.592g

 

 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 |2018-08-14T16:50:12+00:00February 18th, 2015|Categories: E16.5, Mus musculus|Tags: , , , , , |0 Comments
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E16.5 C57BL6 NKX2.1, EMCN, and ACTA2 Confocal Imaging

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

Immunofluorescence- NKX2.1, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL6 E16.5 lungs for NKX2.1, 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-NKX2.1 (R1231) dilute 1:1000. For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, for mouse anti-ACTA2 (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-NKX2.1), 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, 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.

Tissue Used:   

LMM.14.57.4.51 E16.5

Gender: Female

Crown-to-Rump Length: 16.0 mm

Weight=0.574g

 

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 |2018-08-14T16:50:13+00:00February 18th, 2015|Categories: E16.5, Mus musculus|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.