Anti-Semaphorin 4D/CD100 antibody [30/CD100]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Semaphorin 4D/CD100 with ab307685 at 1/5000 dilution (0.1994 ug/ml) followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on human colon. The section was incubated with ab307685 for 30 mins at room temperature, followed by anti-mouse IgG2b antibody (ab190482) for 8 mins during the LeicaDS9800 kit staining procedure. 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1%Triton X-100 permeabilized HDLM-2 (human Hodgkin lymphoma cell) cells labelling Semaphorin 4D/CD100 with ab307685 at 1/100 dilution (9.97 ug/ml), followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing membranous staining in HDLM-2 cell line, no staining was observed in PC-3 cell line. Negative control : PC-3 (PMID : 23775445). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) (Left panel) compared to HDLM-2 (human Hodgkin lymphoma cell) (Right panel) cells labelling Semaphorin 4D/CD100 with ab307685 at 1/1000 dilution (0.1ug) (Red) compared with a Mouse monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti-Mouse IgG (Alexa Fluor® 488, ab150113) at 1/2000 dilution was used as the secondary antibody. Negative control : PC-3 (PMID : 23775445).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Semaphorin 4D/CD100 with ab307685 at 1/5000 dilution (0.1994 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab307685 for 30 mins at room temperature, followed by anti-mouse IgG2b antibody (ab190482) for 8 mins during the LeicaDS9800 kit staining procedure. 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed 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-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Semaphorin 4D/CD100 with ab307685 at 1/5000 dilution (0.1994 ug/ml) followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on rat spleen. The section was incubated with ab307685 for 30 mins at room temperature, followed by anti-mouse IgG2b antibody (ab190482) for 8 mins during the LeicaDS9800 kit staining procedure. 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed 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-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Semaphorin 4D/CD100 with ab307685 at 1/5000 dilution (0.1994 ug/ml) followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on mouse spleen. The section was incubated with ab307685 for 30 mins at room temperature, followed by anti-mouse IgG2b antibody (ab190482) for 8 mins during the LeicaDS9800 kit staining procedure. 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 LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

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Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Blocking buffer : 5% NFDM/TBST. Exposure time : Lane 1, 2 : 15 seconds; Lane 3 : 3 seconds. The expression molecular weight observed is consistent with what has been described in the literature (PMID : 17244710; PMID : 24841081). Band around 25kDa was cleavage fragment(PMID : 17244710). Lysate in Lane 3 was freshly made and used immediately to minimize protein degradation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Observed MW(kDa) 150,120, 25

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (ab307685) at 1/1000 dilution

Lane 1:

Untreated HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

Lane 2:

HDLM-2 whole cell lysate treated with Protein Deglycosylation MIXII at 20 µg

Lane 3:

HDLM-2 whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 96 kDa

false

• WB

Supplier Data

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Blocking buffer : 5% NFDM/TBST. Exposure time : 15 seconds. Negative control : PC-3 (PMID : 23775445). The expression molecular weight observed is consistent with what has been described in the literature (PMID : 17244710; PMID : 24841081). Band around 25kDa was cleavage fragment(PMID : 17244710). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Observed MW(kDa) 150,120, 25

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (ab307685) at 1/1000 dilution

Lane 1:

HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 96 kDa

false

• WB

Supplier Data

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Blocking buffer : 5% NFDM/TBST. Exposure time : Lane 1-3 : 15 seconds; Lane 4 : 3 seconds. Band around 25kDa was cleavage fragment(PMID : 17244710). Observed MW(kDa) 150,120, 25

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (ab307685) at 1/1000 dilution

Lane 1:

Daudi (human Burkitt’s lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 2:

EL4.IL-2 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 3:

Mouse thymus tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 96 kDa

false

• WB

Lab

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (AB307685)

Western blot : Anti-SEMA4D antibody [30/CD100] (ab307685) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab307685 was shown to bind specifically to SEMA4D. A band was observed at 90-120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in SEMA4D knockout cell line. To generate this image, wild-type and SEMA4D knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [30/CD100] (ab307685) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human SEMA4D knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-sema4d-knockout-hct116-cell-line-ab289144'>ab289144</a>)

Lane 2:

SEMA4D knockout HCT 116 cell lysate at 20 µg

Lane 3:

HDLM-2 UNBOILED cell lysate at 20 µg

Lane 4:

PC-3 UNBOILED cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 90-120 kDa

false