Bright field microscope

Paraffin embedded kidneys Sects. (4 μm) were stained with Pico-Sirius Red (PSR), Masson’s Trichrome (MT) and PAS stains respectively. For each sample, six random non-overlapping fields were captured at 200 × magnification using a bright-field microscope (Leica Microsystems, Wetzlar, Germany). The pathological scoring was performed by two independent examiners. For PSR, the area of staining was estimated by Image J software. The final PSR index (COL1 and COL3 abundance) was calculated by multiplying the intensity with the area of staining as per previously described^{26 (link)}. Tubular dilation, interstitial inflammation and fibrosis were assessed by MT staining. Each characteristic of tubular injury was graded on a scale of zero to four based on the percentage of affected area as previously reported^{21 (link)}: Grade 0 = no damage/staining; grade 1 =  < 25% area, grade 2 = 25–50% area; grade 3 = 50–75% area and grade 4 =  > 75% area. Ten non-overlapping fields were assessed, and the scores were averaged. PAS staining was used to score glomerulosclerosis (grade zero to four) based on the level of mesangial expansion and basement membrane thickening as per previously described^{27 (link)}. A total of twenty glomeruli were randomly selected and the whole kidney average glomerulosclerosis index was obtained by averaging scores from all counted glomeruli in one section.

Protein expression of NF-κB p50, TGF-β1, AMPKα, AKT1, and Nrf2 was detected with rabbit primary polyclonal IgG antibodies, whereas primary goat polyclonal IgG antibodies were utilized for detecting KIM-1, in renal tissues (Thermo Fisher Scientific). Following overnight incubation of renal tissue sections with the primary antibodies (1 : 200 concentration; 4°C), the slides were washed and incubated for 30 minutes with ImmPRESS® HRP-conjugated Horse Anti-Rabbit or Anti-Goat IgG Plus Polymer Peroxidase Kits (Vector Laboratories Inc.; CA, USA). Rabbit or goat IgG isotype antibodies (Santa Cruz Biotechnology Inc.; TX, USA) were used in replacement of the primary antibodies to control for nonspecific binding. The slides were then observed with a brightfield microscope (Leica Microsystems, Wetzlar, Germany). Images were then acquired from 10 nonintersecting areas/section. The IHC scores were determined by ImageJ software (https://imagej.nih.gov/ij/), as reported earlier [26 (link)].

Human colon cancer HT29 cells, colon-derived normal fibroblast CCD-18Co cells, lung cancer A549 cells, normal human bronchial epithelial (immortalized with adenovirus 12-SV40 virus hybrid Ad12SV40) BEAS-2B cells, breast cancer MDA-MB-231 cells, normal breast epithelial MCF10A cells, human osteosarcoma U-2OS cells were purchased from ATCC (Manassas, VA, United States). Primary osteoblasts were generated as reported previously (Buondonno et al., 2016 (link)). HMM and HMC were primary cells stabilized in culture as described (Aldieri et al., 2004 (link)). The use of primary cells was approved by Ethical Committee of the San Luigi Gonzaga Hospital, Orbassano, (protocol #126/2016). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Cells were maintained in medium supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin-streptomycin, 1% v/v L-glutamine. Morphological analysis was performed with a bright field microscope (Leica Microsystems, Wetzlar, Germany). At least 5 fields/experimental condition were examined.

To quantify the extent of mucosal damage, the histological analysis was performed. A 0.5 cm segment from the distal colon of mice was immersed in 4% paraformaldehyde (pH 7.4) for 24 h, embedded in paraffin, cut into 4 μm sections using standard histological techniques to prepare paraffin sections (for further analysis, e.g., Immunohistochemistry and immunofluorescence assay). The prepared paraffin sections were stained with hematoxylin and eosin (H&E), observed and photographed with a bright-field microscope (Leica Microsystems, Heerbrugg, Swiss).

Tissues were fixed in 10% formalin for 36 h and embedded in paraffin or frozen-embedded in OCT solution (Tissue-Tek). Paraffin sections and frozen sections were prepared at 4 μm and 12 μm thickness, respectively, and mounted on microscope slides (Trajan Scientific and Medical, VIC, Australia). Ep WAT and liver were stained with Hematoxylin and Eosin (H&E) for histological analysis. For each sample, six random non-overlapping fields were captured at 200× magnification using a bright-field microscope (Leica Microsystems, Wetzlar, Germany). Adipocyte size was measured using Adiposoft, a plugin of the Image J software (https://imagej.net/Adiposoft, January 2019). Adipose tissue morphology was graded from 0 to 4 based on the occurrence of multilocular adipocytes [31 (link)], which reflects the ‘beiging’ level of WAT. The score 0 represents 100% adipocytes being mature with 1 single lipid droplet (unilocular), 1 represents up to 25% of adipocytes being multilocular, 2 represents a 25–50% occurrence of multilocular adipocytes, 3 represents extensive adipose tissue remodeling with 50–75% of adipocytes being multilocular, and 4 indicates that the majority of adipocytes (>75%) are multilocular. Slides were coded and assessed blindly to ensure objectivity. For lipid droplet visualisation in liver tissues, Oil Red O (ORO) staining was used as previously described [9 (link)].

H&E and immunohistochemistry images were captured using a brightfield microscope (Leica Microsystems, Wetzlar, Germany) at different magnifications (10×, 20×, and 40×). Scale bars were added using Image J. All the H&E images and IHC images were assembled as figures using ImageJ, Adobe Illustrator CC (Adobe, San Jose, CA, USA), or MS PowerPoint software.