Postnatal Day 3 C57BL6 ARL13B, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence-ARL13B, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND03 lungs for ARL13B, 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-Arl13b (17711-AP,Proteintech) 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 @ 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-Arl13b), 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.
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 or TB, 3X, 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 in light-sealed box @ 4^oC.

Tissue Used:

LMM.16.83A.4.41

PND03 C57BL6

Gender: Male

Weight= 2.1g

Appendix

Antigen Retrieval Solution

18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5

82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2

1L dH₂O

By kitc5i|2018-08-14T19:12:05+00:00March 22nd, 2017|Categories: Mus musculus, P03|Tags: ACTA2, alpha smooth muscle actin, ARL13B, Emcn, Endomucin, Endothelial Cells, SMA|0 Comments

Postnatal Day 10 C57BL6 ARL13B, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence-ARL13B, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND10 lungs forARL13B, 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.

1. Antigen retrieval, pH 6.0 (times will vary according to microwave).
2. 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).
3. Microwave according to instructions on microwave.
4. Cool on countertop, 15 min.
5. Rinse with dH₂O
6. 1X PBS, 5 min.
7. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
8. For Rabbit anti-Arl13b (17711-AP,Proteintech) 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 @ 4^oC.
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-Arl13b), 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.
10. Store in light-sealed box @ 4^oC.
Tissue Used:
LMM.16.92A.6L.54
PND10 C57BL6
Gender: Male
Weight= 4.1g

Appendix
Antigen Retrieval Solution
18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5
82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2
1L dH₂O
pH 6.0

By kitc5i|2018-08-14T19:14:52+00:00March 21st, 2017|Categories: Mus musculus, P10|Tags: ACTA2, alpha smooth muscle actin, ARL13B, Emcn, Endomucin, Endothelial Cells, SMA|0 Comments

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 03 C57BL6 HOPX, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Gene
Hopx	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas
Emcn	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas
Acta2	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas

Donor Tissue Kindly Provided by Dr. Gloria Pryhuber fromÃ‚Â the University of Rochester Medical Center

Immunofluorescence-HOPX, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND03 lungs for HOPX, EMCN, and ACTA2

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-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 @ 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-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.
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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB or TB, 3X, 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 at 4^o

Tissue Used:

LMM.16.81A.3.34

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|2018-08-18T19:21:33+00:00August 1st, 2016|Categories: Mus musculus, P03|Tags: ACTA2, alpha smooth muscle actin, Emcn, Endomucin, Hopx, SMA|0 Comments

Postnatal Day 10 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/6J PND10 lungs for HOPX, EMCN, and ACTA2

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-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 @ 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-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.
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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB or TB, 3X, 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.16.92A.6L.29

PND03 C57BL6

Gender: Male

Weight= 4.1g

Appendix

Antigen Retrieval Solution

18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5

82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2

1L dH₂O

pH 6.0

By kitc5i|2018-08-14T19:14:55+00:00July 19th, 2016|Categories: Mus musculus, P10|Tags: ACTA2, alpha smooth muscle actin, Emcn, Endomucin, Hopx|0 Comments

Postnatal Day 03 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/6J PND03 lungs for HOPX, EMCN, and ACTA2

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-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 @ 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-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.
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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB or TB, 3X, 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.3.28

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|2018-08-14T19:12:00+00:00July 14th, 2016|Categories: Mus musculus, P03|Tags: ACTA2, alpha smooth muscle actin, Emcn, Hopx|0 Comments

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

The requested content cannot be found

Immunofluorescence- NKX2.1, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND10 lungs for NKX2.1, EMCN, and ACTA2

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-NKX2.1 (R1231) dilute 1:1000, For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, for mouse anti-ACTA2 (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-NKX2.1), 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.
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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB or TB, 3X, 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 at 4^o

Tissue Used:

LMM.16.92A.6L.30

PND10 C57BL6

Gender: Male

Weight= 4.1g

Appendix

Antigen Retrieval Solution

18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5

82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2

1L dH₂O

pH 6.0

By kitc5i|2018-08-14T19:14:56+00:00July 14th, 2016|Categories: Mus musculus, P10|Tags: ACTA2, alpha smooth muscle actin, Emcn, NKX2.1, TTF-1|0 Comments

Postnatal Day 03 C57BL6 NKX2.1, EMCN, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- NKX2.1, EMCN, and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL6 PND03 lungs for NKX2.1, EMCN, and ACTA2

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-NKX2.1 (R1231) dilute 1:1000, 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-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.
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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB or TB, 3X, 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 at 4^oC

Tissue Used:

LMM.16.81A.3.35

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|2018-08-14T19:12:00+00:00July 14th, 2016|Categories: Mus musculus, P03|Tags: ACTA2, alpha smooth muscle actin, Emcn, NKX2.1, TTF-1|0 Comments

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

The requested content cannot be found

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 goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, For rat anti-AGER (MAB 1179-500, RND Systems) dilute 1:100, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4^oC.

Day 2

Rinse slides in PBS-T 3X, 5 min.
Apply secondary antibody, Donkey Alexa Fluor 488 anti-goat IgG (A11055, Lot# 1605893, for anti-EMCN)at 1:200, Donkey Alexa Fluor 568 anti-rat IgG (AB175708 for anti-AGER) 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 or TB, 3X, 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.15.26A.1L.48

C57BL6 PND28

Gender: Male

Weight: 12.4g

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 kitc5i|2018-09-10T20:19:25+00:00April 15th, 2016|Categories: Mus musculus, P28|Tags: ACTA2, AGER, alpha smooth muscle actin, Emcn, Endomucin, Endothelial Cells, SMA|0 Comments

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