Alkaline phosphatase staining kit

ALP activity analysis was carried out utilizing Alkaline Phosphatase Staining Kit (Abcam, ab284936). As directed by the manufacturer, cells were fixed in 95% methanol for 10 minutes, stained with ALP staining reagent for 15 minutes, and then rinsed with wash buffer. Before being treated for 5 minutes with Alizarin Red Solution 2% (Solarbio, G1450), cells were fixed in 95% methanol for 10 minutes. Then cells were rinsed and imaged under a microscope to estimate the extracellular matrix calcification.

Whole calvaria were isolated from two to three-day old murine neonates essentially as we have described (Bost et al., 2001 (link); Gasper et al., 2002 (link); Madrazo et al., 2003 (link)) with the following modifications. Briefly, primary osteoblasts were isolated from whole calvaria through six sequential 15-min trypsin/collagenase P digestions and the cells were maintained in DMEM supplement with 10% FBS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere. At 24 h, primary osteoblasts were plated in 6-well plates at a density of 2 × 10⁵ cells per well and differentiated in αMEM supplemented with 10% FBS, 0.1 M ascorbic acid, 1 M β-glycerol phosphate, and 100 U/mL penicillin/100 μg/mL streptomycin at 37°C in a 5% CO₂ atmosphere. The differentiation media was changed every other day until the experiment/infection at day 10, and the presence of mature osteoblasts was confirmed using an alkaline phosphatase staining kit (Abcam) and positive staining assessed by light field microscopy as we have described (Johnson et al., 2022 (link)).

For histochemical staining (COX/SDH), muscle cross sections on glass slides were incubated in COX medium (100 μM cytochrome c, 4 mM diaminobenzidine tetrahydrochloride, and 20 μg/mL catalase in 0.2 M phosphate buffer, pH 7.0) for 90 min at 37 °C. Sections were then washed in standard PBS, pH 7.4 (2 × 5 min), and incubated in SDH medium (130 mM sodium succinate, 200 μM phenazine methosulphate, 1 mM sodium azide, 1.5 mM nitro blue tetrazolium in 0.2 M phosphate buffer, pH 7.0) for 120 min at 37 °C. Finally, they were washed in PBS, pH 7.4 (2 × 5 min), rinsed in distilled water, and dehydrated in an increasing ethanol series up to 100%, prior to incubation in xylene and mounting in Eukitt (Merck, Burlington, MA, USA). For skeletal muscle fiber-type identification, antibodies against different myosin heavy chain isoforms were used. Anti-myosin heavy chain ‘slow’ antibody was used to identify the MyH7 isoform in type I fibers (Merck, Burlington, MA, USA)). For type IIa fibers, we used myosin A4.74 antibody (obtained from the Developmental Studies Hybridoma Bank, developed by Helen M. Blau, The University of Iowa). For sequential staining we used a diaminobenzidine staining kit as well as an alkaline phosphatase staining kit (both from Abcam, Cambridge, UK).