Biodec r

The calvaria new tissue, together with the surrounding soft and bony tissues were fixed in 4% paraformaldehyde for 3 days. Decalcification was performed in Biodec R (Bio-Optica, Milan, Italy) for 5 days. After dehydration and clarification, samples were included in paraffin. Histological sections of 5 μm thickness were cut and the slides were stained with Goldner’s Masson Trichrome (Bio-Optica). The examination was performed under an optical microscope (Olympus BX43, Miami, FL, USA).
The area of newly formed bone was measured as a calculated as a percentage reported to the total defect area and, as previously shown [20 (link)].

The details of tissue sampling and processing can be found in previous work (De Vreese et al., 2020 [16 (link)]), although not all samples contained the entire ear canal from the ear opening to the middle ear. Briefly, after sampling, specimens of the external ear canal and surrounding tissues were fixed in 10% neutral-buffered formalin and the time from fixation to tissue processing ranged from several days to 18 months. The far-most medial end of the ear canal, together with the TP-complex, was separated and decalcified using a commercial decalcifier (Biodec R, Bio-Optica^®, Milano, Italy). Next, slabs with a diameter of about 3–4 mm were dissected, transverse to the local orientation of the ear canal longitudinal axis, embedded in paraffin, sectioned to a thickness of 4 µm, and mounted on polarized glass slides. Sections for staining with haematoxylin and eosin were obtained from all slabs and dried overnight at 70 °C, followed by automated staining using a Leica Autostainer XL (Leica Biosystems Nussloch GmbH, Milano, Italy). Few sections were stained with Masson’s trichrome with Aniline blue, or Alcian blue according to the standard laboratory protocols. Slides were coverslipped using a mixture of Eukitt^® (ORSAtec GmbH, Bobingen, Germany) and xylene.

After implant removal, bone specimens were immediately fixed in 10% neutral buffered formalin and maintained at room temperature. Then, they were decalcified for at least 72 h in a mixture of formic and hydrochloric acids (BIODEC R, Bio Optica Milano, Italy), sectioned along the longitudinal implant axis and embedded in paraffin. From each bone portion, sections (5 µm) were obtained and stained with hematoxylin-eosin (H&E) for optical microscopy. The following parameters were analyzed in peri-implant tissue corresponding to 2 mm around each implant site: (1) maximum length of the newly formed bone (mm) measured from the implant profile; (2) bone tissue percentage; (3) number of osteoblasts. All data represent the mean of 10 fields.
Furthermore, sections stained with H&E were digitized using the Hamamatsu’s Nanozoomer 2 scanner (Aperio ImageScope, Buccinasco, Milano, Italy). Areas of new bone deposition and fibrous tissue were marked using an imaging computer software (Aperio ImageScope, Buccinasco, Milano, Italy), analyzed according to a protocol proposed by Han, J.-M. et al. [19 (link)], and expressed as total surface area.

The humeral heads were cut, and the obtained fragments were fixed in 4% neutral buffered formaldehyde solution for 48 h, decalcified by Biodec R (Bio-Optica, Milan, Italy) for 6 h, and then routinely processed for paraffin embedding at temperatures not exceeding 56 °C. For immunohistochemical analysis, 6-μm-thick paraffin-embedded tissue sections were deparaffinized and rehydrated with xylene and a graded series of ethyl alcohols (from 100% to 50%), before incubation with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in 0.3% Tween 20 in PBS 1× at room temperature (RT) for 20 min. Sections were then incubated overnight at 4 °C with anti-TGF-β1 antibody (dilution 1:200; GeneTex, Irvine, CA, USA). Antigen was visualized by Envision Dako REAL™ EnVision™ Detection System (Dako, Santa Clara, CA, USA). Sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted in Biomount HM (Bio-Optica, Milan, Italy).

At the end of the experimental period, adult of zebrafish have been carefully euthanized by anesthesia with a dose of 0.7 g/l tricaine methane sulfonate (MS-222) buffered. Gills, liver, gut were excised and immediately fixed in 4% formaldehyde (Bio-Optica) in PBS buffered (Phosphate Buffered Saline, Sigma Life Science) at room temperature for 24 h.
Gills were decalcified, prior to processing, with a decalcifier agent (Biodec R, Bio-Optica) for 2 h at room temperature. Histological examinations were performed according to our standard laboratory procedures. All tissues were washed three times with PBS (0.1 M, pH 7.4), gradually dehydrated in ascending alcohols series (35°, 50°, 70°, 95°, absolute ethanol) for 20 min each one and clarified in xylene for 1 h at room temperature, subsequently embedded in paraffin wax (Medite tissue wax 56–58°C). Five μm thick histological sections cut by microtome (Reichert Jung 1150 Autocut) and collected on glass slides (Menzel Gläser, Thermo Scientific). At least 10 slides of each tissue were collected. The sections were stained with Haematoxylin-Eosin (HE) (Bio-Optica) (Brundo et al., 2011 (link)) and observed under optical microscope (Leica DM750) to identify potential morphological alterations. Photographs were produced using an optical microscope (Leica DMLB) equipped with a digital camera (Leica DFC500).

Samples were fixed in 4% formaldehyde (Bio-Optica) in PBS buffered to 0.1 M, pH 7.4 (Sigma Life Science) at room temperature for 36 h. Gills were decalcified, prior to processing, with a decalcifier agent (Biodec R, Bio-Optica) for 3 h at room temperature. Histological examinations were performed according to our standard laboratory procedures (Brundo et al., 2011 (link); Droutsa et al., 2019 (link)), and processed with Tissue Processing Center TPC 15 Duo (MEDITE^®). The sections were stained with Haematoxylin-Eosin (HE) (Bio-Optica) and observed under optical microscope (Leica DM750, Monument, CO, United States) equipped with a digital camera (Leica DFC500, Monument, CO, United States).