PND7 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- NKX2.1, FOXF1 and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL/6 PND07 mouse lung for NKX2.1, FOXF1 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- NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For goat anti- FOXF1 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 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-FOXF1), 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.15.27A.7L.59

PND07 C57BL6

Gender: Male

Weight: 4.4g

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 dH₂O

pH 6.0

E16.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
E18.5 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 01 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 03 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 07 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 10 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 14 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging
Postnatal Day 28 C57BL6 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

By kitc5i|2018-07-02T13:32:15+00:00February 22nd, 2018|Categories: P07|Tags: ACTA2, FOXF1, NKX2.1|0 Comments

Postnatal Day 7 C57Bl6 NKX2.1, KI67, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence-NKX2.1, Ki-67 and ACTA2

Purpose: Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PN07 lungs for NKX2.1, Ki-67 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-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For mouse anti-Ki-67 (55603, BD Biosciences, Lot# 6251774) 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-NKX2.1), Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-Ki-67) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A (A21136, Lot# 1345055, 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 5X.c
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 at 4^oC.

Tissue Used:

LMM.15.23A.4L.26

C57Bl6 PND7

Gender: Male

Weight: 3.28g

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|2018-09-04T14:43:47+00:00March 28th, 2017|Categories: Mus musculus, P07|Tags: ACTA2, alpha smooth muscle actin, KI67, NKX2.1, TTF-1|0 Comments

Postnatal Day 7 C57BL6 HOPX, AGER, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- HOPX, AGER, and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL6/J PND07 lungs for HOPX, AGER, 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 ^o*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 dH ₂O
1X PBS, 5 min.
Block in 4% Goat serum/PBS-T, 2 hours at RT.
For rabbit anti- HOPX (SC-30216, Lot # C2212, Santa Cruz) 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^o

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-HOPX),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.15.27A.7L.45

C57BL6 PND7

Gender: Male

Weight: 4.4g

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|2018-09-11T17:03:42+00:00April 22nd, 2016|Categories: Mus musculus, P07|Tags: ACTA2, AGER, alpha smooth muscle actin, Hopx, SMA|0 Comments

Postnatal Day 7 C57BL6 HOPX, ABCA3, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- HOPX, ABCA3, and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL6/J PND07 lung for HOPX, ABCA3, 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-HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, for guinea pig anti-ABCA3 (G985, BL 3/10/05, Seven Hills) 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti- HOPX), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-ABCA3) 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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB, 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.15.27A.7L.57

C57BL6 PND07

Gender: Male

Weight: 4.4g

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 dH₂O

pH 6.0

By kitc5i|2018-08-31T01:35:52+00:00April 22nd, 2016|Categories: Mus musculus, P07|Tags: ABCA3, ACTA2, alpha smooth muscle actin, Hopx, SMA|0 Comments

Postnatal Day 7 C57BL6 NKX2.1, SCGB1A1, and TUBB3 Confocal Imaging

The requested content cannot be found

Immunofluorescence NKX2.1, SCGB1A1, and TUBB3

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND07 lung for NKX2.1, SCGB1A1, and TUBB3 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- NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti-TUBB3 (anti-tubulin β 3, cat. # 801201, Biolegend) dilute 1:200 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-NKX2.1), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-SCGB1A1) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A31571, Lot# 1549801, anti-TUBB3) 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.15.27A.7L.32

C57BL6 PND07

Gender: Male

Weight: 4.4g

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 dH₂O

pH 6.0

By kitc5i|2018-09-10T19:55:12+00:00April 22nd, 2016|Categories: Mus musculus, P07|Tags: CCSP, NKX2.1, Scgb1a1, TTF-1, TubB3|0 Comments

Postnatal Day 7 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.15.23A.4L.29

C57Bl6 PND7

Gender: Male

Weight: 3.28g

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|2018-09-10T20:14:55+00:00April 11th, 2016|Categories: Mus musculus, P07|Tags: ACTA2, AGER, alpha smooth muscle actin, Emcn, Endomucin, Endothelial Cells, SMA|0 Comments

Postnatal Day 7 C57BL6 NKX2.1, ABCA3, 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% Goat serum/PBS-T, 2 hours at RT.
For rabbit anti-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, for guinea pig anti-ABCA3 (G985, BL 3/10/05, Seven Hills) 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti- NKX2.1) at 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-ABCA3) 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 15 min.
Wash in PBS-T 3X, 5 min.
Rinse slides in 0.1M PB, 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.46

PND28 C57BL6

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 dH₂O

pH 6.0

By kitc5i|2018-09-11T18:12:37+00:00April 11th, 2016|Categories: Mus musculus, P07|Tags: ABCA3, ACTA2, alpha smooth muscle actin, NKX2.1, SMA, TTF-1|0 Comments

Postnatal Day 7 C57BL6 NKX2.1, AGER, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence: NKX2.1, AGER, and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND07 lung for NKX2.1, AGER, 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- NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, 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, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-NKX2.1),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 incubate 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 proof box @ 4^oC.

Tissue Used: LMM.15.23A.4L.56

C57Bl6 PND7

Gender: Male

Weight: 3.28g

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|2018-08-18T19:37:13+00:00April 11th, 2016|Categories: Mus musculus, P07|Tags: ACTA2, AGER, alpha smooth muscle actin, NKX2.1, SMA, TTF-1|0 Comments

Postnatal Day 7 C57BL6 NKX2.1, VIM, and ACTA2 Confocal Imaging

The requested content cannot be found

Immunofluorescence- NKX2.1, VIM and ACTA2

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND07 mouse lungs for NKX2.1, VIM, 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-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, for goat anti- VIM (SC7557, Lot # C1215) 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 @ 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-VIM ) 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.

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 a light sealed box @ 4^oC.

Tissue Used: LMM.15.27A.7L.30

C57BL6 PND7

Gender: Male

Weight: 4.4g

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 dH₂O

pH 6.0

By kitc5i|2018-08-18T19:39:05+00:00April 11th, 2016|Categories: Mus musculus, P07|Tags: ACTA2, alpha smooth muscle actin, NKX2.1, SMA, TTF-1, VIM, Vimentin|0 Comments

Postnatal Day 7 C57BL6 SOX9, SFTPC, and ACTA2 Confocal Imaging

The requested content cannot be found

Gene
Sox9	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas
Sftpc	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas
Acta2	LungGens	Ensemble	Genecards	NCBI	The Human Protein Atlas

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-SOX9 (AB-5535, Lot # 2167153, Millipore) dilute 1:100, For goat anti-SFTPC (SC-7706, Santa Cruz) 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-rabbit IgG (A21206, Lot# 1608521, for anti-SOX9), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti-SFTPC) 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, 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.

Tissue Used:

LMM.15.23A.4L.40

PND7 C57BL6

Gender: Male

Weight: 3.3g

By kitc5i|2017-05-18T12:41:10+00:00May 1st, 2015|Categories: P07|Tags: ACTA2, alpha smooth muscle actin, pro-SPC, Sftpc, SOX9|0 Comments

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