Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (human malignant melanoma melanoma) cells labeling SIRP alpha with ab260039 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in SK-MEL-28 cells Negative control : Jurkat (PMID : 17982459) is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260039).

• Flow Cyt

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Flow Cytometry - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

Flow cytometric analysis of Jurkat (Human T cell leukemia T lymphocyte, Left) / THP-1 (Human monocytic leukemia monocyte, Right) cells labeling SIRP alpha with ab260039 at 1\600 compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

Negative control : Jurkat.

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 (ab260039).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling SIRP alpha with ab260039 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human cerebrum (PMID : 9062191) is observed. The section was incubated with ab260039 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

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

Heat mediated antigen retrieval was performed 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 (ab260039).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling SIRP alpha with ab260039 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human spleen is observed. The section was incubated with ab260039 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with hematoxylin.

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

Heat mediated antigen retrieval was performed 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 (ab260039).

• Flow Cyt

Lab

Flow Cytometry - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab260039).

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab260039 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 mins on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab260039 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/2,500 dilution)) for 30 mins on ice. The cells were simultaneously stained with CD14.

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

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on CD14+.

• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR22930-163] - BSA and Azide free (AB267401)

Western blot : Anti-SIRPA antibody [EPR22930-163] (ab260039) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab260039 was shown to bind specifically to SIRPA. A band was observed at 75-85 kDa in wild-type A549 cell lysates with no signal observed at this size in SIRPA knockout cell line. To generate this image, wild-type and SIRPA knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR22930-163] (<a href='/en-us/products/primary-antibodies/sirp-alpha-antibody-epr22930-163-ab260039'>ab260039</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SIRPA knockout A549 cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

SW480 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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