Orai1K14 and Orai2−/− mice were perfused with 4% paraformaldehyde, and mandibles were isolated, decalcified (10% EDTA) for 2 weeks, and embedded in paraffin to obtain thin sections (5 μm thick). Immunofluorescence staining was performed using an antigen retrieval step as described (69). The following primary antibodies (all rabbit-raised) were incubated at 4°C overnight: polyclonal rabbit anti-ORAI1 (1:75; S.F., New York University Medical Center) and monoclonal rabbit anti-ORAI2 [1:75; Abcam; clone EPR10043(2)]. The next day, sections were washed twice for 5 min each with phosphate-buffered saline before incubation for 1 hour with biotin-labeled anti-rabbit immunoglobulin G (1:500 dilution; Vector Laboratories) and washing and incubation with streptavidin Alexa Fluor 488 (1:800 dilution; Life Technologies). Samples were embedded using Fluoromount mounting medium (Novus) containing DAPI (Thermo Fisher Scientific). Images were obtained using a Nikon Eclipse 2000TE or a Leica TCS SP5 II confocal microscope and edited using Icy BioImage analysis.

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