ab27853 staining PLVAP/PV-1 in b.End3 cells. The cells were fixed with 4% paraformaldehyde (10 minutes), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated overnight at 4°C with ab27853 at 2 µg/mL and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150165, Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor® 488), pre-absorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 minutes).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

IHC image of PLVAP/PV-1 staining in a section of frozen normal mouse large intestine performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab27853, 1 µg/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

IHC image of PLVAP/PV-1 staining in a section of frozen normal mouse brain performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab27853, 1 µg/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. The inset secondary-only control image is taken from an identical assay without primary antibody. Mouse brain tissue is a negative control tissue, showing no staining of the primary antibody.

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.

Flow cytometry overlay histogram showing bEND.3 (mouse brain endothelioma cell line) cells stained with ab27853 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab27853) (1x10⁶ in 100 µL at 1 µg/mL) for 30 min on ice.

The secondary antibody Goat anti-rat IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150165) was used at 1/2000 for 30 min on ice.

Isotype control antibody (black line) was Rat IgG2aκ (ab18450) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.