Anti-B7H4 antibody [EPR23665-20]

• Flow Cyt

Lab

Flow Cytometry - Anti-B7H4 antibody [EPR23665-20] (AB252438)

Flow cytometry overlay histogram showing left SKBR3 positive cells and right negative HeLa stained with ab252438 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interactionfollowed by the antibody (ab252438) (1x 106 in 100μl at 10.0 μg/ml (1/50)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

• Flow Cyt

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Flow Cytometry - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

• mIHC

Lab

Multiplex immunohistochemistry - Anti-B7H4 antibody [EPR23665-20] (AB252438)

Fluorescence multiplex immunohistochemical analysis of the human breast (Formalin/PFA-fixed paraffin-embedded sections). Panel A : merged staining of anti-B7H4 (ab252438, red; Opal™690), anti-CD10 (ab255609, gray; Opal™520) and anti-FABP4 (ab92501, cyan; Opal™570) on human breast. Panel B : anti-B7H4 stained on glandular lumens. Panel C : anti-CD10 stained on myoepithelial cells. Panel D : anti-FABP4 stained on adipocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The section was incubated in three rounds of staining : in the order of ab252438 at 1/100 dilution (4.69 μg/ml), ab255609 at 1/1000 dilution (0.615 μg/ml) and ab92501 at 1/10000 dilution (0.047 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

• IP

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Immunoprecipitation - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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).

All lanes:

Immunoprecipitation - Anti-B7H4 antibody [EPR23665-20] (ab252438)

Predicted band size: 31 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

• WB

Lab

Western blot - Anti-B7H4 antibody [EPR23665-20] (AB252438)

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

All lanes:

Western blot - 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 31 kDa

Observed band size: 45 kDa,65 kDa

false