Dm500b microscope

Following lung fixation, lung sections were analyzed for histology. Lung sections were stained with H&E and Masson’s Trichrome (collagen deposition). Randomly selected areas (five fields) from 5-μm thick lung sections were captured at 100× (H&E) and 200× (Masson’s Trichrome) magnification using a Leica DM500B microscope (Leica, Germany). Large airways and vessels were avoided for the lung morphometry. To measure mean linear intercept (MLI), a grid with parallel lines spaced at 60 μm was overlaid onto the image, and the length of each chord, defined by the intercept with alveolar walls, was recorded. The degree of collagen deposition was measured using ImageJ software and expressed as percentage of collagen deposition per total septal area. The lung microvasculature was determined by counting the number of von Willebrand factor (vWF)-positive vessels (diameter <50 μm) in five random images at 100× magnification, and the number of macrophages in lung tissue was counted by measuring CD68-positive cells in five random high fields. Tissue sections were incubated with primary antibody (vWF, 1:400; CD68, 1:200, Abcam). Afterward, slides were washed and incubated with Alexa Fluor 488-congugated donkey anti-rabbit (1:500) antibody.

At 7 and 14 dpi, lung tissues were fixed in 4% paraformaldehyde and then dehydrated, embedded in paraffin, and cut into 5 μm-thick sections. The sections were stained with hematoxylin and eosin (H&E) and Masson’s Trichrome using a Leica DM500B microscope (Leica, Germany).

To visualize apoptosis, TUNEL staining was performed according to the manufacturer’s instructions using the Promega DeadEnd™ Fluorometric TUNEL System # G3250 [14 (link), 22 (link)]. The fluorescein-12-dUTP-labeled DNA was visualized by Leica DM500 B Microscope and digital camera. DAPI nuclear counterstaining was performed to visualize total cells. Osteocyte and PDL apoptotic fluorescent cells were analyzed using a custom ImageJ macro. Raw images were converted to binary images using the Intermodes threshold, and individual nuclei were counted using the built in Analyze Particles function. TUNEL-positive cells were counted on the compression side of alveolar bone and PDL (mesial surface of the distobuccal root) and normalized per number of DAPI-positive cells in the same tissue section. The ratio of cells was calculated by one observer for each section as TUNEL-positive cells/total cells in both the AB and PDL. The ratio from three sections was then averaged for each mouse. Sixteen randomly selected measurements were repeated with an intra-class coefficient of r = 0.9998. The averaged values were used to conduct statistical analysis.