Immunohistochemical analysis of paraffin-embedded mouse breast carcinoma tissue labeling B7H4 with ab252438 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).Positive staining on mouse breast cancer cells. The section was incubated with ab252438 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse breast tissue labeling B7H4 with ab252438 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).Positive staining on mouse breast. The section was incubated with ab252438 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

All lanes : Anti-B7H4 antibody [EPR23665-20] (ab252438) at 1/1000 dilution

Lane 1 : SK-BR-3 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2 : MDA-MB-468 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3 : NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell), whole cell lysate at 10 µg
Lane 4 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 31 kDa
Observed band size: 45,65 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

B7H4 is N-glycosylated protein. The level of B7H4 glycosylation varies between different cells and tissues. The bands at 65kDa and 45kDa represent glycosylated B7H4. (PMID: 15878339).

Negative control: HT-29 and HeLa. (PMID: 15878339). 

Exposure time: 3 minutes

B7H4 was immunoprecipitated from 0.35 mg SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate with ab252438 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252438 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: SK-BR-3  whole cell lysate 10 ug

Lane 2: ab252438 IP in SK-BR-3 whole cell lysate 10 ug

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252438 in SK-BR-3 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 127 seconds

The bands at 65kDa represent glycosylated B7H4 (PMID: 15878339).

Flow cytometric analysis of SK-BR-3 (Human breast adenocarcinoma epithelial cell) cells labelling B7H4 with ab252438 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 647, ab150079) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T+OE-612 cells labelling B7H4 with ab252438 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and membranous staining in HEK-293T cell line transfected with HA-tagged B7H4 expression vector. ab236632 Anti-HA tag rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling B7H4 with ab252438 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast carcinoma (PMID: 15756008). The section was incubated with ab252438 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labeling B7H4 with ab252438 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human ovarian carcinoma (PMID: 27439899). The section was incubated with ab252438 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling B7H4 with ab252438 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast (PMID: 27439899). The section was incubated with ab252438 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins