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 (link)). 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 (link))]. 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.
Protocol detail
Immunofluorescence Analysis of Orai1 and Orai2 in Mandibles
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Alexa fluor 488
Antibodies
Antigen
Biotin
Clone
Confocal microscope
Dapi
Dilution
Edta
Embedded paraffin
Immunofluorescence
Immunoglobulin g
Mandibles
Mice
Novus
Paraformaldehyde
Phosphate
Rabbit
Saline
Streptavidin
Thin sections
Vector
Corresponding Organization : New York University
Other organizations : Carl Zeiss (United States), Dalhousie University, New York University Abu Dhabi
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Variable analysis
independent variables
Orai1 K14 Orai2 −/−
- Immunofluorescence staining of ORAI1 and ORAI2 proteins
- Perfusion with 4% paraformaldehyde
- Decalcification in 10% EDTA for 2 weeks
- Embedding in paraffin to obtain thin sections (5 μm thick)
- Antigen retrieval step
- Primary antibodies: polyclonal rabbit anti-ORAI1 (1:75) and monoclonal rabbit anti-ORAI2 [1:75]
- Secondary antibody: biotin-labeled anti-rabbit immunoglobulin G (1:500 dilution)
- Streptavidin Alexa Fluor 488 (1:800 dilution)
- Embedding using Fluoromount mounting medium containing DAPI
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