Axioskop 40

Sections were stained with Harris hematoxylin (Newprov, Pinhais, Paraná, Brazil) for 40 s, washed under running tap water for 2 min and stained with Masson's trichome solution for 17 min. Images of consecutive regions of the wound area were taken in × 200 magnification using an optic microscope (Axioskop 40 Zeiss; Carl Zeiss). The identification of collagen fibers was made by deconvolution of color images on ImageJ software Version 1.52 (NIH, Bethesda, Maryland, USA). During color image deconvolution, images with Masson’s trichrome stain were deconvolved, and the green component was identified as the collagen fibers. The area of the green collagen fibers was measured using the “threshold” tool. The threshold was manually adjusted until the entire green area was highlighted in red. Then, the threshold was measured as the percentage of the stained area⁵⁸ .

The mice were euthanized after 12 h, 3, and 7 days (Sup. Fig. S1f). Samples were collected 2.0 mm beyond the wounds and fixed in paraformaldehyde 4% for 48 h and processed for both optimal cutting temperature compound or paraffin embedding. Optimal cutting temperature compound was done in 20 µm cryosections (CM3050 S, Leica Microsystems, Morrisville, North Carolina, USA), and the slides were observed by confocal microscopy. Paraffin blocks were serially sectioned using a microtome at 6 μm sections (Leica). Five consecutive histological fields in five different sections were selected to analyze re-epithelialization and inflammatory process grading (40 × or 100 ×). According to a previous study^{57 (link)}, the inflammatory process intensity, fibrin clot formation, degree of angiogenesis, epithelium differentiation, and indentation were given a score of 0 to 5 based on its level of abundance in the wound beds according to a previous study. The granulation tissue area, epithelium area and thickness were quantified by the Fiji software (NIH). All samples were analyzed with an optical microscope (Axioskop 40 Zeiss, Göttingen, Lower Saxony, Germany), and one blinded examiner undertook all histological analysis. Detailed staining protocols are described below.

Immunohistochemistry was performed for α-SMA detection at day 7 post-surgery in the wound sections. Briefly, the slides were deparaffinized and dehydrated, followed by incubation with 3% hydrogen peroxide and 1% bovine serum albumin (BSA). The sections were then incubated with a polyclonal rabbit anti-mouse α-SMA primary antibody (MM1, Novocastra, Newcastle, UK, 1:200) at room temperature for two hours. The immunolabeling was visualized through incubation in 3,3-diaminobenzidine (DAB) solution (Dako, Carpinteria, California, USA). Then, the sections were stained with Mayer’s hematoxylin and covered. Negative controls were obtained by omission of the primary antibody, which was substituted for 1% PBS-BSA. Images of 10 consecutive fields were taken in × 200 magnification using an optic microscope (Axioskop 40 ZEISS; Carl Zeiss, Gottingen, Germany). Immunostaining for α-SMA was quantified by color deconvolution plugin on ImageJ Version 1.52 (NIH). The red component was identified as α-SMA staining, and a threshold was manually adjusted and finally measured as the percentage of the stained area in the wound beds.

Periodontal tissue samples from periodontal pockets or healthy oral mucosa extracted during surgery of impacted third molars were fixed in 10% buffered formalin, embedded in paraffin wax, and cut longitudinally (3 μm). The sections were deparaffinized, rehydrated, and stained with H&E for evaluation of the inflammatory infiltrate. Inflammatory cells were counted in four fields in two independent sections, using a light microscope (Axioskop 40 ZEISS; Carl Zeiss, Gottingen, Germany) at 400x magnification. Data were expressed as total inflammatory cells/field.

Sirius red staining is a method used to access the presence of eosinophils^{60 (link)}. Briefly, slides were incubated in Harris hematoxylin for 2 min, rinsed in tap water, and then 100% ethanol. Subsequently, slides were immersed in an alkaline (pH 8–9) Sirius Red solution (CI 35780, Sigma Aldrich) for 2 h. Ten random foci were selected from low power in an optic microscope (Axioskop 40 Zeiss; Carl Zeiss) and were assessed by one blinded examiner. Eosinophils were counted per viewing field (× 400 magnification) and averaged for each wound bed.