Nanozoomer 2.0 ht system

Mouse femur bones were isolated and fixed in 10% neutral buffered formalin for 24 hours. Bones were decalcified in 14% EDTA for 14 days at 4^oC, embedded in paraffin and sectioned 5μm thick at the histology core of the Washington University Musculoskeletal Research Center. Standard H&E technique was used for all bone sections. Images were collected using the Olympus NanoZoomer 2.0-HT System, Alafi Neuroimaging Laboratory. Immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded slides as previously described (32 ). Slides were stained with the following antibodies: anti-IL-6 primary antibody (ab6672, 1:100, AbCam), pMK2 primary antibody (3007, 1:50, Cell Signaling), Total p38 primary antibody (9212, 1:100, Cell Signaling), Biotinylated Donkey anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody (Cat#:31821, 1:500, 2.2μg/ml, Thermofisher).

Adipose and liver tissues were fixed in 10% buffered formalin, dehydrated by an ethanol gradient, and stored in 70% ethanol at 4°C before paraffin embedding. Liver and adipose tissue sections were incubated with hematoxylin and eosin or Masson’s trichrome, stain. Elastin protein deposition was visualized by incubating adipose tissue sections with anti-Elastin antibody, as described previously [17 ]. Images were captured using an Olympus Nanozoomer 2.0-HT System with NDP.scan 2.5 imaging software. For adipose tissue adipocyte size and number, ImageJ software was used to make measurements in four fields per section.

Tissues were fixed in 10% buffered formalin, dehydrated by an ethanol gradient, and stored in 70% ethanol at 4°C before paraffin embedding. Tissue sections were stained with hematoxylin and eosin. Images were taken using an Olympus Nanozoomer 2.0-HT System with NDP.scan 2.5 image software.