Eukitt

Explanted sample sections were stained with Masson trichrome according to the manufacturer’s protocol (Bio-Optica, Italy, Milan). Tissue slides were stained also with toluidine blue. Samples were deparaffinized in xylene and dehydrated using a graded series of ethanol. Then, sections were placed in water for 5 min and transferred to toluidine blue for 4 min, blotted carefully. Sections were embebbed in absolute alcohol and subsequently cleared in xylene, mounted on a glass slide using Eukitt (Bio-Optica, Italy, Milan). Metachromatically stained mast cells were enumerated by counting 5 high-power fields (40×) per section using Axiovision Zeiss (Milan, Italy) microscope.

Skin sections were deparaffinized in xylene and dehydrated by a graded succession of ethanol, 5 min in each solution [82 (link)]. The sections were next placed in water for 5 min, relocated to toluidine blue for 4 min, and then blotted cautiously. Sections were positioned in absolute alcohol for 1 min, cleared in xylene, and fixed on glass slides using Eukitt (Bio-Optica, Milan, Italy). The number of metachromatic stained mast cells was obtained by counting 5 high-power fields (40×) per section using a DM6 microscope (Leica, Milan, Italy).

To assess the degree of myelination/demyelination process, staining with LFB stain kit (Abcam, Waltham, MA 02453, USA # ab150675) was performed in the deparaffinized sections following manufacturer’s instructions. Briefly, spinal cord sections were incubated in LFB solution at 56 °C O/N, then washed in 95% alcohol. Subsequently, the sections were incubated in lithium carbonate solution and 70% ethyl alcohol and, finally, counterstained in the cresyl violet solution. After dehydration, the sections were assembled with Eukitt (Bio-Optica, Milan, Italy) and observed by light microscopy (AxioVision, Zeiss, Milan, Italy). The slides were analyzed by a pathologist blinded to the treatment groups. Images were taken focusing on the perilesional area of SCI and shown by using objective lens at 10× and 20× magnification.

Mast cells identification was assessed in the palm of paw sections. Briefly, for evaluation of number of mast cells, tissue sections were stained with toluidine blue. Sections were deparaffinized in xylene and dehydrated through a graded series of ethanol, 5 min in each solution. The sections were next placed in water for 5 min, transferred to toluidine blue for 4 min and then blotted carefully. Sections were placed in absolute alcohol for 1 min, cleared in xylene, and mounted on a glass slide using Eukitt (Bio-Optica, Milan, Italy). Sections were stained blue and the mast cells were stained purple. Metachromatically stained mast cells were enumerated by counting five high-power fields (40×) per section using Axiovision Zeiss (Milan, Italy) microscope.

Haematoxylin and eosin staining was performed on 7 μm-thick cryosections fixed with 4% paraformaldehyde for 10 min at 4 °C. The staining was performed according to standard protocols. For Milligan’s trichrome staining, sections were fixed for 1 h with Bouin’s fixative (Sigma-Aldrich) and rinsed for 1 h under running water. Sections were then rapidly dehydrated to 95% EtOH in graded ethanol solutions, successively passed in 3% potassium dichromate (Sigma-Aldrich) for 5 min, rapidly washed in distilled water, stained with 0,1% acid fuchsin (Sigma-Aldrich) for 30 s, washed again in distilled water, passed in 1% phosphomolybdic acid (Sigma-Aldrich) for 3 min, stained with Orange G (2% in 1% phosphomolybdic acid) (Sigma-Aldrich) for 5 min, rinsed in distilled water, passed in 1% acetic acid (VWR) for 2 min, stained with 1% Fast Green for 5 min (Sigma-Aldrich), passed in 1% acetic acid for 3 min, rapidly dehydrated to 100% EtOH and passed in Xylene before mounting with Eukitt (Bio-Optica).

A morphological and a histochemical stains were used to process the slides [4 (link), 6 (link)]. Slices were then rehydrated using progressive alcohol solutions (from 100 to 30% ethanol), to distilled water after being deparaffinized in xylene [17 (link)],G. [75 (link)], subsequently, they were stained using Mallory trichrome and AB/PAS [3 (link)]. After staining slides were mounted using Eukitt (BioOptica Milano S.p.A, Milan, Italy, Europe). Data on the stains used in this study are included in Table 1.Table 1

Stains used in this study

Stain name	Type	Catalogue number	Supplier
Mallory trichrome	Morphological	04–020,802	BioOptica Milano S.p.A, Milan, Italy, Europe
Alcian blue/Periodic Acid Schiff (AB/PAS)	Histochemical	04–163,802	BioOptica Milano S.p.A, Milan, Italy, Europe

Embryos at the indicated developmental stage were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. Where indicated tadpoles were irradiated with 5 Gys with Faxitron before fixation. After fixation, embryos were embedded in 1% LMP agarose and then in paraffin with Diapath automatic processor (Diapath). To assess histological features hematoxylin/eosin staining (Diapath) was performed according to standard protocol and samples were mounted in Eukitt (Bio-Optica). For IHC analysis, paraffin was removed with xylene and the sections were re-hydrated in graded alcohol. Antigen retrieval was carried out using pre-heated target retrieval solution for 45 min. Tissue sections were stained with 0.3% H₂O₂ for 10 min at room temperature for quenching of endogenous peroxide activity, and then blocked with 0.2% Fetal Bovine Serum (FBS) in PBS supplemented with 1% BSA for 1 h and incubated for 2 h with primary antibodies. Mouse monoclonal anti-phospho-histone H2A.X (Ser139) (JBW301, Millipore, 1:500) was used for staining. Antibody binding was detected using a polymer detection kit (GAR-HRP and GAM-HRP, Microtech), followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab). All sections were counterstained with Mayer's hematoxylin and visualized using a bright-field microscope.