Aperio at turbo digital pathology scanner

Tumor tissues were embedded in paraffin after fixation in 10% formalin before IHC staining. Four‐µm‐thick sections were de‐waxed, rehydrated with graded alcohols, and then mounted on glass slides. Following PBST (phosphate buffered saline containing 0.05% Tween‐20) rinses, the slides were incubated with the primary antibodies for 1 h and then incubated with appropriate HRP‐conjugated secondary antibodies at room temperature for 30 min. The slides were washed with PBST, incubated with DAB (3, 3′‐diaminobenzidine), and then scanned with an Aperio AT Turbo Digital Pathology Scanner (Leica Biosystems). IF staining was performed on tumor tissue slides by fixing, permeabilizing, blocking with 1% BSA, and incubating with antibodies overnight at 4 °C. Nuclei were counterstained with DAPI before CLSM imaging. Table S1, Supporting Information, illustrates the antibodies used.

Femurs from 3‐ and 7‐month‐old male and female mice were isolated and fixed in 4% paraformaldehyde solution and decalcified using 10% EDTA for 2 weeks. Samples were then dehydrated using a graded ethanol series and were paraffin‐embedded. Coronal sections (5 μm) were obtained using the Leica RM 2255 automated microtome (Leica Biosystems, Wetzlar, Germany), and stained for alkaline phosphatase (ALP) or tartrate‐resistant acid phosphatase (TRAP), for osteoblast and osteoclast visualization, respectively. High‐resolution slide scans were acquired using the Aperio At Turbo Digital Pathology Scanner (Leica Biosystems). For each randomly numbered sample, 3 to 5 sections were analyzed in full for bone cell numbers, at 4× magnification, using the ImageJ software (NIH). Undecalcified bones were embedded in methyl methacrylate (MMA), and 1‐μm sections were cut on an ultramicrotome. These sections were stained with the Goldner trichrome method as previously described.⁽²²⁾ All histological analyses were conducted as per the recommendations of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee.⁽²³⁾