Recombinant Alexa Fluor® 488 Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab199091)

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab198978 (red line). The cells were fixed with 80% methanol (5 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 (ab198978, 1/500 dilution) for 30 min at 22ºC.

Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 488 (ab199091) used at the same concentration and conditions as the primary antibody. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab199091 using various fixation and permeablisation. The cells were fixed, washed and permeablised as indicated below;

4% formaldehyde (10 min, room temperature)/0.1% PBS-Triton X-100 (15 min, room temperature)

80% methanol (5 min, -20°C)/0.1% PBS-Triton X-100 (15 min, room temperature)

4% formaldehyde (10 min, room temperature)/90% methanol (30 min, -20°C)

The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab199091) for 30 min at 22ºC at the following concentrations - Blue line (Unlabelled), Black line (0.1μg/ml), Red line (1μg/ml) and Green line (10μg/ml) .

Acquisition of >5,000 events were collected using a 50mW Blue laser (488nm) and 530/30 bandpass filter.

Immunofluorescent analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells, fixed with 4% formaldehyde (10 min). The cells were permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab199091 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control) at 1/500 dilution (showing no signal) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (pseudocolored in red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 100% methanol (5min).

Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/.

CRT was measured using dual staining of PI and a monoclonal CRT antibody. Following 24 h after incubation, cells were washed with PBS, detached with 200 µL of accutase, and washed twice with 2 mL of FACS buffer (500 mL sheath fluid + 2 g bovine serum albumin + 1 g NaN₃ in 100 mL H₂O). Each sample was split into two vials and one was stained with monoclonal primary rabbit anti‐CRT antibody (Abcam, ab196158) while the other was stained with rabbit IgG, monoclonal isotype control (Abcam, ab199091) for 40 min at 4°C. Cells were then washed once with FACS buffer. 0.5 µL of PI was added to each sample immediately before being quantified with a flow cytometer. Fifteen thousand events were collected and only the PI− cells were analyzed for surface CRT expression. Data were analyzed and gated using the FlowJo software (FlowJo LLC, version 10). Data were expressed as percent CRT positive after accounting for nonspecific binding with their corresponding isotype.