Flow Cytometry - GC1q R antibody [60.11] (ab24733)Image courtesy of David Sanchez Martin by Abreview.
ab24733 used in Flow Cytometry. U937 cells were cultured in RPMI 10% FCS, treated with PMA (5nM) and, 6-12 hours later, cells were harvested, spinned and resuspended to 200,000 cells in 100µl. ab24733 used at 10µg/ml for 30 minutes at 4°C. A phycoerythrin conjugated goat anti-mouse polyclonal was used as the secondary antibody at a 1/100 dilution.
Flow Cytometry - Anti-GC1q R antibody [60.11] (ab24733)
Overlay histogram showing HeLa cells stained with ab24733 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab24733, 0.5μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150117) at 1/4000 dilution for 30 min at 22°C.
Isotype control antibody (black line) was mouse IgG1 [15-6E10A7] (ab170190, 0.5μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GC1q R antibody [60.11] (ab24733)
IHC image of ab24733 staining in human tonsil formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab24733, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Immunocytochemistry/ Immunofluorescence - Anti-GC1q R antibody [60.11] (ab24733)
ICC/IF image of ab24733 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab24733 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
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