Vs desktop virtual slide system

Tissue sections from the paraffin embedded blocks were used for the histological and immunohistochemical study. H and E staining was done on SAT, EPI, and BAT (Details described in Data S1) for the measurement of adipocyte size. The measurements of adipocyte size in SAT and EPI depots were made after imaging an entire hematoxylin stained section (n = 3/group), in an automated slide scanner (Olympus). The imaging software, VS‐DESKTOP Virtual Slide System (Olympus), was used to randomly select four areas across tissue sections and the diameter of 50 cells in each area was determined. For irregularly shaped adipocytes, a line was drawn across the maximum diameter. For Picrosirius Red Staining (PSR), tissues slides were first stained in hematoxylin for 10 min, followed by 3–5 dips in acid alcohol, blueing in Scotts water for 30 s, then stained with Picrosirius Red for 1 h and washed in two changes of acidified water (Junqueira et al., 1979 (link); Puchtler et al., 1973 (link)). Immunohistochemical staining of SAT and EPI was done for the collagen after retrieving antigen by heating slides in a microwave oven in Tris‐EDTA buffer pH 9.0 for 10–15 min (detail in Data S1).

Adipose tissue fixed in 10% neutral buffered formalin was used for H and E staining, Picrosirius Red Staining (PSR) and immunohistochemical study as described previously [40 (link)]. H and E staining was used to measure the adipocyte size after imaging the entire hematoxylin stained section (n = 3/group), using an automated slide scanner (Olympus, Tokyo, Japan) and the imaging software, VS-DESKTOP Virtual Slide System (Olympus). PSR staining was used to detect collagen deposition in adipose tissue. Immunohistochemical staining for collagen VI (1:500; Catalog number ab6588, Abcam) was done after antigen retrieval by heating slides in a microwave oven. Secondary antibody (1:200, Biotinylated anti-rabbit IgG, catalog number BA-1000; Vector Laboratories^®, Burlingame, CA, USA) was used followed by the addition of an avidin-biotin complex (Vectastain ABC kit, catalog number PK-4000; Vector Laboratories^®) followed by 3, 3-Diaminobenzidine (DAB) (catalog number K3468; Dako^®, Santa Clara, CA, USA).