H&E and other immunohistochemistry

AT were fixed in 10% neutral buffered formalin for at least 48 hours, trimmed, routinely processed for histology, sectioned at 4-μm thickness, and stained with hematoxylin and eosin.

Immunohistoc hemical staining (IHC) for the macrophage marker CD68 was performed on unstained 4-mm thick sections of the AT. The primary antibody was a mouse monoclonal antibody (clone ED1. Serotec, Oxford, UK) at a dilution of 1:5 000 with an incubation period of 60 min. Multiple tissues from a domesticated brown rat (Rattus norvegicus) were used as controls.

Photomicrographs of each sample were acquired using a camera (Olympus DP70) mounted on a light microscope (Olympus BX41) with a commercial software program (cellSens, Olympus Corporation, Tokyo, Japan). Up to 13 photomicrographs were captured for each sample of AT at 200× and 400× magnification at a resolution of 4080×3072 pixels.

Up to 12 photomicrographs of each sample were randomly selected for quantitative analyses using a commercial software program (Photoshop CS6, Adobe Systems Inc., San Jose, CA). In sections of the AT, the number of positive-staining cells for CD68 was recorded for each selected photomicrograph at 200× magnification, and the mean was calculated per sample. Similarly, the surface areas of 10 randomly-selected adipocytes from each group were measured at 400× magnification, and the mean was calculated per sample.