Mayer s hematoxylin

For lipid staining, 3 μm frozen-kidney sections were fixed with 10% formalin for 5 min, rinsed with distilled water, immersed in 100% 1,2-propanediol (catalog 398039, Sigma Alderich, St. Louis, MO, USA) for 5 min and stained with Oil Red O for 9 min at 60 °C. The sections were subsequently immersed in 85% 1,2-propanediol for 5 min and washed with distilled water. Then, the sections were stained for 3 min with Mayer’s hematoxylin (Bio Optica), washed with distilled water and mounted with an aqueous solution. Twenty-five fields per animal were randomly acquired using bright-field microscopy (ApoTome). Fiji ImageJ Software (http://imagej.net/Fiji (accessed on 13 September 2021)) was used for the quantification of the number of lipid droplets per field (original magnification ×630).

Immunohistochemistry staining on peritoneal sections was conducted using the following antibodies: polyclonal rabbit anti-TGF-b (MBS462142, Mybiosource, USA) at 1:100 dilution, TNF- (ab6671, Abcam, UK) at 1:100 dilution, and VEGF (LS-B7747, LSBio, USA) at 1:100 dilution in the current investigation. The sections were deparaffinized, rehydrated, and treated in a target retrieval solution of Tris–EDTA (pH = 9). To deliver unmasked antigens, samples containing the target retrieval solution were put in a 98 °C heating bath and kept there for 20 min. To halt endogenous peroxidase, samples were treated with hydrogen peroxide (3 percent H2O2) in PBS for 15 min, and normal rabbit serum (5 percent) in PBS was used to avoid nonspecific background staining. The samples were incubated with primary antibodies for one hour. Secondary antibody staining using a goat antirabbit biotinylated antibody (prediluted, Biocare, USA) for 20 min was used to identify primary antibodies. Then, 20-min incubation with prediluted streptavidin horseradish peroxidase (sHRP) (Biocare, USA) was done. Finally, DAB was used as a chromogen to observe antibody binding areas, and Mayer's Hematoxylin (Bio Optica, Italy) was used to counterstain the samples. IHC grading was performed on a scale of 0 to 3 (0: no reaction ǀ 1: mild reaction ≤ 10% ǀ 2: moderate reaction 10–30% ǀ 3: intensive reaction ≥ 30%).

After washing with phosphate buffer sulphate (PBS) 0.1 M pH 7.4, cell cultures were fixed for ten min with 10% neutral buffered formalin as previously described^{28 (link),29 (link)}. Cells were washed with sterile double-distilled water and subsequently stained with a filtered ready to use Oil Red O solution (Bio-Optica, Milano, Italy) for twenty min at room temperature. The cells were then washed with sterile double-distilled water and stained with Mayer's Hematoxylin (Bio-Optica, Milano, Italy) ready-to-use solution for 1 min at room temperature and then washed again with sterile double-distilled water. Slides were treated with Aqueous Mount Quick Medium (Bio-Optica, Milano, Italy). Cells were observed in an EVOS FL Auto Cell Imaging System with EVOS Onstage Incubator (Thermo Fisher Scientific, USA) photo microscope, at 200 × and 400 × magnification. The images were analyzed using ImageJ software 1.51n version (NIH, Bethesda, MD, USA) to count the cells within ten representative fields (200 × magnification, number of cells expressed on mm2) and to calculate the area of fifty randomly chosen cells (200 × magnification; area expressed in μm2).

To assess the mature adipocytes’ formation after 21 days of adipogenic differentiation, the presence of lipid droplets was analyzed by Oil Red O staining. LDSCs and ADSCs cells were fixed in 10% neutral buffered formalin (NBF), washed, incubated for 3 min in 60% isopropanol and then Oil Red O solution was applied for 15 min. After washing, cells were counterstained with Mayer’s hematoxylin (Bio-Optica, Milan, Italy) for 1 min and imaged. Quantification of lipid droplets was performed by dissolving the Oil red O dye in 100% isopropanol and measuring absorbance at 450 nm on multichannel spectrophotometer (Multiskan Ascent plate reader, ThermoLab Systems, Helsinki, Finland).

MVECs were seeded onto glass coverslips, grown to 70% confluence, fixed with 3.7% buffered paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Slides were then washed, treated with 3% H₂O₂ in PBS for 15 minutes at room temperature and subsequently blocked with Ultra V block (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes. Cells were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (catalog number ab181373; Abcam) at 1:20 dilution in 1% BSA in PBS, followed by incubation with biotinylated secondary antibodies and streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) at room temperature.
Immunoreactivity was developed with 3-amino-9-ethylcarbazole (AEC kit; LabVision). Irrelevant isotype-matched and concentration-matched rabbit IgG (Sigma-Aldrich) were used as negative controls. Nuclei were counterstained with Mayer’s hematoxylin (Bio-Optica). Immunolabeled cells were examined with a Leica DM4000 B microscope (Leica Microsystems) and photomicrographs were captured with a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).

Human muscle tissue was harvested in formalin, fixed, paraffin embedded and sections were prepared as previously described [44 (link)]. Formalin-fixed paraffin-embedded consecutive sections (3 µm thickness) were dewaxed, hydrated by graded decrease alcohol series and stained for histological analysis. The following antibodies were used for immunohistochemical staining (IHC): mouse anti-MURF1, dilution 1:100 (C11, Santa Cruz Biotechnology), mouse anti-PAX7, dilution 1:100 (Developmental Studies Hybridoma Bank, Iowa, IA, USA), rabbit anti-CD31, dilution 1:20 (LSBio, Seattle, Washington, USA). Nuclei were counterstained by hematoxyling according to standard protocol (Mayer’s Hematoxylin, #05-06002/L, Bio Optica, Milano, Italy). Representative images were acquired using a THINDER Imager 3D Tissue microscope (Leica, Wetzlar, Germany) with LAS X Navigator software. The entire slide was scanned at 20× magnification to select CD31 and MurF1 hotspots within five random circular grids of 100 μm diameter. Each CD31-and MurF1-positive signal was manually counted. To quantify PAX7 positive cells, 15 random fields were evaluated at 40× magnification.