The left lungs embedded in paraffin were serially sectioned at a thickness of 4-μm for standard H&E staining, immunohistochemistry, and immunofluorescence. Pulmonary vessels with external diameters smaller than 150 μm were selected. Medial thickness was determined as follows: percent medial thickness (% MT) = (circumferenceext/π − circumferenceint/π)/(circumferenceext/π) × 100%. The circumferenceext and circumferenceint were demarcated by the external and internal elastic lamina, respectively. Images of pulmonary vessels were captured with a Nikon microscope digital camera system, and circumferences were measured with the image analysis program.
Microscope digital camera system
Manufactured by Nikon
Sourced in Japan
The Nikon microscope digital camera system is a high-quality imaging solution designed for use with Nikon microscopes. The system captures detailed digital images of microscopic samples, allowing for precise documentation and analysis.
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Lab products found in correlation
Anti α sma, by Merck Group (2 mentions)
Rabbit anti perk1 2 antibody, by Cell Signaling Technology (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Tunel apoptosis assay kit, by Beyotime (1 mentions)
12 protocols using microscope digital camera system
1
Measurement of Pulmonary Vascular Remodeling
2
TXA Effects on Spinal pERK Expression
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To measure the effect of TXA on spinal dorsal horn with phosphorylated extracellular signal-regulated kinase (pERK), spinal cord slices were perfused with Krebs solution for at least 3 h before TXA application, after which TXA (1 mM) was applied in the perfusate for 10 min. After drug treatment, the slices were fixed in 4% paraformaldehyde for 60 min, equilibrated with sucrose overnight, cut on a cryostat at a thickness of 16 μm, and mounted on slides. Sections were incubated with rabbit anti-pERK1/2 antibody (Cell Signaling Technology, Danvers, MA; 1:1,000) for 2 days at 4 °C. The sections were then incubated with biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA; 1:400) for 4 h at room temperature. Sections were then processed with a Vectastain ABC system kit (Vector Laboratories) following the manufacturer’s instructions. To determine the mean number of pERK-positive neurons in the superficial laminae (I–II), at least five nonadjacent sections were randomly selected, and cells were counted under a microscope-digital camera system (Nikon, Tokyo, Japan).
3
Lung Histology and Imaging Protocol
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After hemodynamic measurements and exsanguination, the left lungs were fixed for histology in 10% neutral buffered formalin, and the right lungs were snap-frozen in liquid nitrogen. H&E and immunofluorescence (IF) staining experiments were conducted as previously described [33 ]. Nuclei were labeled by DAPI staining. Images were captured with a Nikon microscope digital camera system.
4
Immunostaining of α-SMA in Cells
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Cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, permeabilized with 0.2% Triton X100 in PBS for 10 min, blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), and incubated with anti-α-SMA (1:100, Sigma-Aldrich, St. Louis, MO, USA) antibodies overnight at 4°C. Cells were washed and incubated with Alexa Fluor® 594-conjugated goat anti-mouse IgG (Beijing Zhong Shan-Golden Bridge Biological Technology Company, Beijing, China) for 30 min in the dark. After washing with PBS three times, the slices were mounted with 4’,6-diamidino-2-phenylindole (DAPI, Beijing Zhong Shan-Golden Bridge Biological Technology Company, Beijing, China) lysis buffer. Immunofluorescence was observed under a microscope digital camera system (Nikon, Tokyo, Japan).
5
Transwell Migration and Invasion Assay
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Cell migration assays were performed using 8.0 μm pore transwell chambers (Millipore, Billerica, MA, USA). Cells were treated with 5 ng/ml TGF-β1 or 100 ng/ml FSTL1 protein for 24 h and seeded in the upper chamber of the transwells (1 × 105 cells/chamber). FBS-containing DMEM medium was placed in the bottom chambers and used as a chemoattractant. After incubation for 6 h at 37°C, the cells that had migrated through the 8.0 μm membrane pore were counted. Specifically, the upper surface of membrane was swabbed with a cotton swab to remove noninvasive cells and washed with PBS. Invasive cells on the bottom surface were fixed in 4% paraformaldehyde for 30 min and quantified in high-power fields using a microscope digital camera system (Nikon, Tokyo, Japan), after staining the nuclei with DAPI. Invasion assays were performed using similar procedures, coating the transwell chambers with 50 μl of Matrigel (BD Biosciences, San Jose, CA, USA), and allowing it to solidify for 30 min at 37°C before performing the assay.
6
Detecting Apoptosis in Mouse Lungs
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Mouse lungs were fixed, embedded and sectioned into 5 μm thick slices. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed using TUNEL Apoptosis Assay Kit (C1086, Beyotime, Beijing, China) according to the manufacturer’s instruction. Nuclei were labeled by DAPI staining. Images were captured with a Nikon microscope digital camera system.
7
Quantifying Pulmonary Vessel Medial Thickness
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The left lungs of the mice were embedded in paraffin, serially sectioned at a thickness of 4 μm then stained using haematoxylin and eosin (H&E) and immunostaining. With the aid of the H&E stain, all pulmonary vessels with external diameters <100 μm in entire left lung were selected and the medial thickness of these vessels quantified by immunofluorescent labelling with anti-α-SMA (1:200 dilution, Sigma-Aldrich, USA), visualised with second layer Fluor (AF) 488-labelled goat anti-mouse IgG (ZsBio, China). Medial thickness was calculated as a percentage of the external diameter of the vessels as follows: percentage medial thickness (%MT) = (external diameter − internal diameter)/external diameter × 100 [17 (link)]. The data were segregated into four groups based on the external diameters of the vessels: 0–25, 26–50, 51–75 and 76–100 μm. Images of pulmonary vessels were captured with a Nikon microscope digital camera system and circumferences measured using its image analysis software as previously described [17 (link)].
8
Cardiac Ventricular Remodeling Measurement
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Heart tissue was removed. The RV free wall was dissected from the left ventricle (LV) and ventricular septum (S). The RV hypertrophy index (RVHI) was calculated by the wet weight ratio of the RV to the LV plus S using the formula: RV/(LV + S) × 100%. The ratio of RV weight to body weight (BW) was also examined.
Cardiomyocyte cross-sectional diameter was determined in the RV and LV as measures of cardiac ventricular tissue remodeling. Four-micron sections of the RV and LV were stained with hematoxylin and eosin (H&E), and the diameter of individual cardiomyocytes in histological sections of the left and right ventricular walls was measured. Microscopic images were analyzed in a blinded manner using a Nikon microscope digital camera system and its image analysis program (Nikon, Tokyo, Japan).
Cardiomyocyte cross-sectional diameter was determined in the RV and LV as measures of cardiac ventricular tissue remodeling. Four-micron sections of the RV and LV were stained with hematoxylin and eosin (H&E), and the diameter of individual cardiomyocytes in histological sections of the left and right ventricular walls was measured. Microscopic images were analyzed in a blinded manner using a Nikon microscope digital camera system and its image analysis program (Nikon, Tokyo, Japan).
9
Cardiac Ventricle Remodeling in Mice
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The method was described in previous papers [14 (link), 15 (link)]. Mice were anesthetized with 2% pentobarbital (50 mg/kg, i.p.). The mice were then sacrificed, and the hearts were collected. To evaluate the extent of right ventricle (RV) hypertrophy, RV tissues were separated and the weights of the RV and the left ventricle (LV) plus interventricular septum (S) were measured respectively. The right ventricular hypertrophy index (RVHI) was defined as: RVHI (%) = [RV/(LV + S)] × 100. The ratio of RV weight to body weight (BW) was also calculated.
Cardiomyocyte cross-sectional diameter was determined in the RV as measures of cardiac ventricle tissue remodeling. 4 μm sections of the RV were stained with H&E. Microscopic images were analyzed in a blinded manner using Nikon microscope digital camera system and its image analysis program (Nikon, Tokyo, Japan).
Cardiomyocyte cross-sectional diameter was determined in the RV as measures of cardiac ventricle tissue remodeling. 4 μm sections of the RV were stained with H&E. Microscopic images were analyzed in a blinded manner using Nikon microscope digital camera system and its image analysis program (Nikon, Tokyo, Japan).
10
Localization of Salusin-β in Aorta
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The thoracic aorta was rinsed with cool sterile phosphate buffered saline (PBS). Paraffin-embedded heart and blood vessel sections (5 μm) were permeabilized using 0.1% Triton X-100 for 10 min after deparaffinization and rehydration. The sections were washed in PBS then blocked with 10% goat serum for 1 h, then incubated with rabbit anti-salusin-β antibody overnight at 4°C, and were then visualized under a Nikon microscope digital camera system (Nikon, Tokyo, Japan).
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