Neutral buffered formalin

Skeletal muscle samples were fixed in 10% neutral buffered formalin (Bio-Optica, Milan, Italy), and, after overnight washing, were embedded in paraffin as previously described [17 (link)]. The samples were placed in the cassettes in longitudinal and cross directions after wax infiltration. Tissue samples (4–5 μm) were cut from paraffin blocks by a rotary manual microtome (Leica RM2235, Milan, Italy) and then mounted on silane-coated slides (Menzel-Gläser, Braunschweig, Germany) and preserved at room temperature. Afterwards, the sections were dewaxed in xylene, hydrated by graded ethanol, and stained by Hematoxylin and Eosin staining for histological evaluation, muscle fibers identification, detection of structural alterations, and histomorphometric measurements. The slides were examined with a Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany), and pictures were taken with a digital camera (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany).

Target IVDs with adjacent vertebrae were collected 2-weeks’ post-injury and were fixed in 10% neutral buffered formalin (Bio-Optica, Milan, Italy) for 1 week at room temperature. Tissue was decalcified in an EDTA-glycerol solution and was processed for paraffin embedding. Sequential transversal 5-µm sections of the IVD were collected. Sections were deparaffinized in xylene, rehydrated through a graded series of ethanol, and processed for alcian blue/picrosirius red staining, immunofluorescence for collagen type II, and immunohistochemistry for CD68 and CD3.

NB specimens were fixed for 24–48 hr in 10% neutral buffered formalin (Bio Optica) and paraffin embedded. For histological analysis, 4 μm thick serial sections were stained with Harris’ hematoxylin and 1% eosin (Bio Optica). For immunohistochemistry experiments, serial sections were treated in a microwave oven three times for 5 min in citrate buffer (pH 6.0) at 750 W, then subjected to 3% H₂O₂ for 15 minutes and saturated with 20% goat serum in PBS-T, for 1 hr at 37°C. Samples were then incubated overnight at 4°C in a humidified chamber with the specific primary antibody diluted in PBS-T as follows: anti-VEGFA (1:50), anti-PHD3 (1:100), anti-PDK1 (1:100), anti-HIF-1α (1:150), and anti-N-Myc (1:200). Negative controls were made with PBS-T alone. After incubations with the biotinylated secondary antibody and the HRP-streptavidin (LSAB2 System-HRP kit, Dako), the reactions were developed and visualized as described above. The slides, which were blinded for specimen identification, were evaluated by a pathologist in a randomized order using a bright field microscope. Specimens were evaluated for the percentage of positive cells and for the staining intensity. The percentage of positive cells was confirmed by ImmunoRatio, a free, automated web-based image analysis application for scoring immune-stained slides [37 (link)]. (https://dx.doi.org/10.17504/protocols.io.jtxcnpn)

Only for Case 1, it was possible to carry out morphopathologic analysis. The animal was slaughtered in strict accordance with the European slaughter regulations (CE n° 1099/2009), and the internal and external genital organs were observed for the detection of pathologic lesions or abnormalities.
Samples from testis and reproductive tissue were collected and preserved in 10% neutral buffered formalin (code no. 05-01007Q, Bio-Optica, Milan, Italy), dehydrated and embedded in paraffin (code no. 06-7920, Bio-Optica, Milan, Italy). Tissue sections were stained with haematoxylin and eosin (HE) for morphological analysis.

Explants were fixed in 10% v/v neutral-buffered formalin (Bio Optica Milano S.p.A., Milan, Italy) for at least 7 days. The samples were washed in deionized water, and the areas of interest (areas that contained the inserted implant) were cut using an electric autopsy saw and decalcified for at least 4 days in an EDTA-based solution (Milestone™, Shelton, CT, USA). The decalcification status was checked daily. Samples were then histologically processed in an automated system and embedded in paraffin. Then, 4–5 µm sections in silane-coated slides were taken from each bone specimen using a microtome (ThermoScientific™, Waltham, MA, USA) and stained with hematoxylin and Eosin (ThermoScientific™, Waltham, MA, USA). Tissue sections were dewaxed in xylene and hydrated through decreasing concentrations of alcohol until 100% water for 5 min. Samples were then incubated in hematoxylin (Richard-Allan Scientific™, Waltham, MA, USA) for 5 min, washed and differentiated, incubated in alcoholic eosin (Richard-Allan Scientific™, Waltham, MA, USA) for 2 min, dehydrated, and mounted. Representative slices from the previous osteomyelitis defect and the interface between bone and biomaterials were analyzed, and the presence of inflammation (acute and chronic), bone necrosis, and new bone formation were evaluated.

Specimens for histology were fixed in 10% Neutral Buffered Formalin (Bio-Optica, Milan, Italy) and then embedded in paraffin; 5 μm slides were cut and placed in glass slides for histology. Sections were deparaffinized in xylol, rehydrated in graded alcohol series (Sigma Aldrich, Milano, Italy), then washed in water and stained with Hematoxylin (BioOptica) and Eosin (BioOptica) (H&E) for histological examination. The slices were analyzed on an Olympus 41microscope.

The ovaries were collected immediately after the sacrifice and fixed in 10% neutral buffered formalin (Bio-Optica, Milano, Italy) over night. The ovaries were processed and serially sectioned as previously described (21 (link)). Briefly, slices were stained with haematoxylin and eosin (DDK Italia, Vigevano, Italy) and finally analyzed under light microscopy to assess follicles diameter and morphological features (28 (link)). To select a representative follicular population, one slice every 150 μm was considered, and were used to identify the antral follicles to be analyzed. Follicle diameter was calculated as the mean distance between opposite basal membrane portions, whereas the wall thickness was calculated as the sum of theca interna and granulosa cell layers. According to follicle diameter, the follicular population was divided in two classes: early-small antral follicles (150–300 μm) and large antral follicles (>300 μm) (29 (link)–31 (link)). The presence of morphological cystic features (32 (link)–36 (link)) was recorded for each antral follicle. Two investigators independently and blindly performed morphological analysis.