Immunocytochemistry/ Immunofluorescence - Syntaxin 6 antibody [3D10] (ab12370)This image is courtesy of an anonymous Abreview
ab12370 at 1/200 staining a mouse macrophage cell line by ICC/IF. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 24 hours. A FITC conjugated donkey anti-mouse antibody was used as the secondary.
Syntaxin 6 was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 10µg of Mouse monoclonal to Syntaxin 6 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab12370. Secondary: Protein G-HRP at 1/500 dilution. Band: 25kDa:Syntaxin 6.
Overlay histogram showing PC12 cells stained with ab12370 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab12370, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1](ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.