Postnatal Day 3 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND03 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
For Rabbit anti-CSPG4 (AB5320, Millipore) 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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
Apply secondary antibody, Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1387792, for anti-CSPG4) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, 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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in light-sealed box @ 4^oC.

Tissue Used:

LMM.16.81A.3.26

PND03 C57BL6

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 kitc5i|2018-08-14T19:12:05+00:00March 20th, 2017|Categories: Mus musculus, P03|Tags: ACTA2, alpha smooth muscle actin, CSPG4, Emcn, NG2, Pericytes|0 Comments

Postnatal Day 10 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND10 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
For Rabbit anti-CSPG4 (AB5320, Millipore) 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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
Apply secondary antibody, Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1387792, for anti-CSPG4) Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, 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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in light-sealed box @ 4^oC.

Tissue Used:

LMM.16.84A.5L.20

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 kitc5i|2018-08-14T19:14:53+00:00March 20th, 2017|Categories: Mus musculus, P10|Tags: ACTA2, alpha smooth muscle actin, CSPG4, Emcn, NG2, Pericytes|0 Comments

Postnatal Day 14 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND14 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
For Rabbit anti-CSPG4 (AB5320, Millipore) 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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).

Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in light sealed box @ 4^oC

Tissue Used: LMM.15.34A.5L.55

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

By kitc5i|2018-08-19T23:19:24+00:00April 6th, 2016|Categories: Mus musculus, P14|Tags: ACTA2, alpha smooth muscle actin, CSPG4, Emcn, Endomucin, Endothelial Cells, NG2, Pericytes, SMA|0 Comments

Postnatal Day 14 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 PND14 lungs for CSPG4, NKX2.1, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Goat serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in a light sealed box @ 4^oC.

Tissue Used: LMM.15.33A.6L.52

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

By kitc5i|2018-08-19T23:20:04+00:00April 4th, 2016|Categories: Mus musculus, P14|Tags: ACTA2, alpha smooth muscle actin, CSPG4, NKX2.1, Pericytes, SMA, TTF-1|0 Comments

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

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND01 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in a light sealed box @ 4^oC.

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

By kitc5i|2018-08-17T23:27:50+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: ACTA2, alpha smooth muscle actin, CSPG4, Emcn, Endomucin, Endothelial Cells, NG2, Pericytes, SMA|0 Comments

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

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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Goat serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.

Store slides in light sealed box @ 4^oC

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

By kitc5i|2018-08-17T23:16:41+00:00April 1st, 2016|Categories: Mus musculus, P01|Tags: ACTA2, alpha smooth muscle actin, CSPG4, NKX2.1, Pericytes, SMA, TTF-1|0 Comments

Postnatal Day 28 C57BL6 CSPG4, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND28 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in a light sealed box @ 4^oC.

Tissue Used:

LMM.15.20A.1.31

PND28 C57BL6

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 6.0J

By kitc5i|2018-08-14T19:25:46+00:00April 12th, 2015|Categories: Mus musculus, P28|Tags: ACTA2, CSPG4, Emcn, Endomucin, NG2, Pericytes|0 Comments

Postnatal Day 7 C57Bl6 CSPG4, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- CSPG4, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND07 lungs for CSPG4, EMCN, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Donkey serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in a light sealed box @ 4^oC.

Tissue used

LMM.15.23A.4L.45

PND7 C57BL6

Gender: Male

Weight: 3.3g

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 kitc5i|2018-08-18T19:56:44+00:00April 12th, 2015|Categories: Mus musculus, P07|Tags: ACTA2, alpha smooth muscle actin, CSPG4, Emcn, NG2, Pericytes|0 Comments

Postnatal Day 7 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 PND07 lungs for CSPG4, NKX2.1, and ACTA2 with antigen retrieval.

Day 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.
Antigen retrieval, pH 6.0 (times will vary according to microwave).
10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
Microwave according to instructions on microwave.
Cool on countertop, 15 min.
Rinse with dH₂O
1X PBS, 5 min.
Block in 4% Goat serum/PBS-T, 2 hours at RT.
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 @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
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.
Rinse in PBS-T 3X, 5 min.
Dilute DAPI 1:2000 and apply to slides for 10 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB 3X for 5 min.
Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
Allow Prolong Gold to cure overnight at room temp in light sealed box.
Store slides in a light sealed box @ 4^oC.

Tissue Used: LMM.15.17A.5.24

PND7 C57BL6

Gender: Male

Weight= 4.00g

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 kitc5i|2018-08-18T19:57:34+00:00April 12th, 2015|Categories: Mus musculus, P07|Tags: ACTA2, alpha smooth muscle actin, CSPG4, NG2, NKX2.1, Pericytes|0 Comments

Gene	LungGENS	Ensemble	Genecards	NCBI	Human Protein Atlas	Wikipedia

FN1	Human Day 1	Ensemble	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	Lung Proteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Foxf1	Human Day 1	Ensemble	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Abca3	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Gene	LungGENS	Ensemble	Genecards	NCBI	Human Protein Atlas	Wikipedia

FN1	Human Day 1	Ensemble	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	Lung Proteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Ager	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Abca3	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Gene	LungGENS	Ensemble	Genecards	NCBI	Human Protein Atlas	Wikipedia

Krt14	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	Lung Proteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Muc5b	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Tuba4a	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Gene	LungGENS	Ensemble	Genecards	NCBI	Human Protein Atlas	Wikipedia

Syp	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	Lung Proteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Sox2	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Tubb3	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Gene	LungGENS	Ensemble	Genecards	NCBI	Human Protein Atlas	Wikipedia

Fn1	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	Lung Proteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Foxf1	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

Acta2	Human Day 1	Ensembl	Genecards	NCBI	The Human Protein Atlas	Wikipedia
	Human 20 Months
	Human Neonate to Adult
	LungProteomics Human 20 Months
	Mouse Lung Proteomics E16.5-P28

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