Axostar plus

RHE sections were collected for histological evaluation using standard haematoxylin and eosin (H&E) staining. In brief, RHE tissues were fixed with 10% neutral formalin, embedded in paraffin, and sectioned at 7 μm. Subsequently, sections were deparaffinized with xylene and then stained with haematoxylin and eosin, as previously described [34 (link)]. All sections were evaluated using an AxioVision microscope (Axostar Plus equipped with Axio-Cam MRc, Zeiss, GE, Germany), and the histological results are shown at 20× magnification (50 μm of the Bar scale).

Immunohistochemical localization was executed as previously described [34 (link),36 (link)]. In brief, all slides were incubated overnight with the following primary antibodies: anti-ZO1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-33725), anti-occludin (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-133256), anti-IL-1β (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-32294), anti-IL-2 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-133118), and anti-IL-12 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-7925). After washing with PBS, sections were incubated with a secondary antibody for 1 h at room temperature. The reaction was revealed by a chromogenic substrate (DAB). The images were acquired using an optical AxioVision microscope ((Axostar Plus equipped with Axio-Cam MRc, Zeiss, GE, Germany). For immunohistochemistry, the images are shown at 20× magnification (50 μm of the Bar Scale).

To identify epidermal thickness and inflammatory cells infiltration, sections were stained with hematoxylin–eosin (H&E). Histological evaluations were performed as previously described by Reference [11 (link)].
After dehydration in graded ethanol and xylol, the tissues were embedded in paraffin and cut to the microtome in order to obtain sections of 7 μm thickness. Tissue sections, after being deparaffinized in xylol and rehydrated through a descending scale of ethanol, were stained with hematoxylin–eosin (H&E, Bio-Optica, Milano, Italy) and examined with an optical microscope (Axostar Plus equipped with Axio-Cam MRc, Zeiss, New York, NY, USA) to observe the structure of the epidermis. The histological results were shown at 10× (100 μm of the bar scale).

Histological evaluation was performed as previously described by Paterniti et al. [49 (link)]. Tumor samples were quickly removed and fixed with 10% buffered formalin for at least 24 h at room temperature. After dehydration in graded ethanol and xylene, tumor samples were embedded in paraffin and sectioned at 7 μm thickness. After staining with hematoxylin and eosin, sections were observed by an optical microscope (Axostar Plus equipped with Axio-Cam MRc, Zeiss, GE, Germany). The histological results are shown at 20× and 40× magnification (bar scale at 50 and 20 μm, respectively). All histological analyses were executed in a blinded manner.

Golgi impregnation was performed according to the directions supplied by FD NeuroTechnologies, FD (FD NeuroTechnologies, Ellicott City, MD, USA). Blocks of spinal cord tissue were placed directly into solutions A and B, without rinsing for 2 weeks in the dark at room temperature. Forty-eight hours after placing the blocks in solution C (4°C), they were frozen on dry ice and stored at −70°C until sectioned. Cryostat sections (100 μm) were cut at −25°C and mounted onto gelatinized slides. Slides were allowed to dry in the dark, and the rest of the staining process performed as previously described (Wallace et al., 2006 (link)). Neurons chosen for tracing met the following criteria: (1) completely impregnated with Golgi stain, (2) unobscured by other impregnated neurons or precipitant, (3) 70% of the dendritic tree was visible within the plane of focus, and (4) neurons must have been located in the outer one-half of the granule cell layer in the dentate gyrus. Cells chosen for analyses had to be well-impregnated, clearly distinguishable from adjacent cells and have continuous unbroken dendrites. Spines were counted under oil (X100), using light microscopy (Axostar Plus equipped with Axio-Cam MRc, Zeiss), and the entire visible dendritic length measured by an imaging computer program (Axio-Vision, Zeiss). Spine density was calculated referring to the length of the dendrite.