Non-Abcam antibody was used: Invitrogen Rabbit Anti Goat AF555 Host species: Rabbit Clonality: Polyclonal Conjugation: Alexa Fluor® 555
Macrophage cell line Mono-Mac6 was grown in RPMI/10% FCS and grown on glass cover slips in 6 well plates. Fixed in 1%PFA prior to staining. Wash 3x3min in PBS. Block 10% rabbit serum for 20min at RT. Primary Ab diluted 1/500 in PBS and incubated overnight at 4deg, also works by incubating for 2 hours at RT. Wash 3x3min in PBS. Secondary - rabbit anti-goat AF555 (red) 1/1000 in PBS for 1h at RT. Wash 3x3min in PBS Counterstained with Hoechst 33258 (blue) for 10mins at RT. Wash 3min in dH2O Coverslips removed tapped dry and placed on glass slides and mounted in Invitrogen ProLong gold and kept at 4deg until imaged.
Abcam user community
Submitted May 25 2012
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