Vs120 l100 virtual slide microscope

The stained slides were scanned at 20-X magnification using the Olympus VS120–L100 Virtual Slide Microscope (Tokyo, Japan). Images were quantified using ImageJ (NIH) by 2 independent examiners blinded to the groups. For each sample, 5 images (of 5 fields) were used for quantification and the average positive area normalized to the total stained area (as percentage) was used for statistical analysis.

To investigate the expression of SGLT-2 in paraffin-embedded renal (or cardiac) sections, immunofluorescence imaging was performed on kidneys from all groups as previously described [78 (link)]. Sections from kidneys were incubated for 45 min at 70 degrees, dewaxed with xylene washes and hydrated by graded ethanol washes of 100% (twice for 3 min), 95%, 80% and 70% followed by water immersion (twice for 4 min). Next, slides were steamed for 45 min with 1× citrate Antigen Retrieval Buffer (ab93678). Sections were rinsed with PBS, permeabilized with 0.2% triton x-100 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min blocking with 10% donkey serum (45 min) and incubated overnight with primary antibody Anti-SGLT2 (sc-393350) in blocking solution. On the next day after wash, the slides with PBS were incubated for 1 h with conjugated secondary antibodies: Donkey anti-Mouse IgG, Alexa Fluor 555 (ThermoFisher Scientific A-10037; 1:400). Nuclei were stained with DAPI before mounting with ProLong Gold Antifade (Invitrogen P36934) and coverslips. For quantification, slides were scanned at 20× magnification using the Olympus VS120–L100 Virtual Slide Microscope (Tokyo, Japan) and five images from randomly selected heart sections (10 magnification) were used to quantify SGLT2 using Image J.

Briefly, tumor tissue was fixed in 4% paraformaldehyde overnight and embedded in paraffin. Sections (4 μm) were cut using a microtome (Leica) and mounted on glass slides. Samples were de-paraffinized and heat antigen retrieval was performed prior to immunostaining with antibodies specific for human CEA (Roche in house), human CD3 (Abcam, Cat No.: ab5690), and human PD-L1 (Ventana, Cat No.: 790-4905). Sections were counterstained with hematoxylin (Sigma Aldrich) and slides were scanned using Olympus VS120-L100 Virtual Slide Microscope. Quantification of percentage of positive stained tumor area for PD-L1 or CEA was performed in whole scans with Definiens software. Raw data was transferred to GraphPad software for analysis of significance. A total of five mice per treatment group was evaluated.