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Postnatal Day 28 Mouse Lung Second Harmonic Imaging

Postnatal Day 28 mouse lungs were inflation fixed with 4% PFA and incubated overnight at 4oC. After overnight fixation, lungs were washed several times DEPC-treated 1X PBS and dehydrated through a Methanol (MeOH) series to 100% MeOH. Lung tissue was then washed in 3 changes of 100% MeOH for 30 minutes. Once tissue is equilibrated in 100% MeOH, remove air bubbles by squeezing out the air bubbles or by using a vacuum.

Clear in BA:BB 1:2  (1 part benzyl alcohol: 2 parts benzyl benzoate). For a 1.5cm x 1.5 cm x 5mm piece of tissue, it takes ~ 1 hour to clear.

Transfer lungs to fresh BA:BB. Image on a multiphoton microscope with glycerol immersion objectives. Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample. We use either a 10X NA 0.5 WD 5.5mm or a 20X NA 1.0 WD 8.2mm.  Image using backscattered SHG with GaAsP NDDs using 840nm excitation and 420/20 filter for SHG, 525/50 for autofluorescence and a 585/50 for pigment (pigment, carbon particles and other debris will “explode” under IR excitation and needs to be captured to remove from final datasets). Mount tissue in BA:BB in 3D printed sample holders and use a coverglass bottom dish as a lid for the sample. The coverglass bottom dish is filled with glycerol to match the refractive index of the sample.

By |2018-08-14T19:25:38+00:00February 23rd, 2018|Categories: Mus musculus, P28, Uncategorized|0 Comments

PND28 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

PND28 NKX2.1, FOXF1, and ACTA2 Confocal Imaging

Immunofluorescence-  NKX2.1, FOXF1 and ACTA2

 

Purpose:  Stain slides of 7µm frozen sections of C57BL/6J PND28 mouse lung for NKX2.1, FOXF1 and ACTA2 with antigen retrieval.

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96oC. *We usually do a series of three runs (6-7 minutes each run) to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  5. Microwave according to instructions on microwave.
  6. Cool on countertop, 15 min.
  7. Rinse with dH2O
  8. 1X PBS, 5 min.
  9. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
  10. For rabbit anti- NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For goat 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 @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. 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.
  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.
  1. Store slides in light sealed box @ 4oC

Tissue Used:   

LMM.15.24A.6L.75

PND28 C57BL6

Gender: Male

Weight: 10.5g

 

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

Related Experiments

By |2018-08-14T19:25:48+00:00February 22nd, 2018|Categories: Mus musculus, P28|Tags: , |0 Comments

Postnatal Day 28 C57Bl6 NKX2.1, KI67, and ACTA2 Confocal Imaging

Postnatal Day 28 C57Bl6 NKX2.1, KI67, and ACTA2 Confocal Imaging

Immunofluorescence-NKX2.1, Ki-67 and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL/6J PND28 lungs for NKX2.1, Ki-67 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-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 @ 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-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.
  3. Rinse in PBS-T 5X.c
  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 at 4oC.

 

Tissue Used:   

LMM.15.26A.1L.28

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

Related Experiments