Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free

• Flow Cyt

Lab

Flow Cytometry - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling B7H4 with ab252438 at 1/1000 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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• Flow Cyt

Unknown

Flow Cytometry - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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 2.5 ug/ml 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 2 ug/ml dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

• IP

Unknown

Immunoprecipitation - Anti-B7H4 antibody [EPR23665-20] - BSA and Azide free (AB272707)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252438).

All lanes:

Immunoprecipitation - Anti-B7H4 antibody [EPR23665-20] (<a href='/en-us/products/primary-antibodies/b7h4-antibody-epr23665-20-ab252438'>ab252438</a>)

Predicted band size: 31 kDa

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