Anti-CD66b antibody [RM1323]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [RM1323] (AB325777)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD66b with ab325777 at 1/20000 (0.025 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human spleen.

The primary antibody was incubated 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 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [RM1323] (AB325777)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD66b with ab325777 at 1/20000 (0.025 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil.

The primary antibody was incubated 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 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [RM1323] (AB325777)

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling CD66b with ab325777 at 1/20000 (0.025 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human gastric cancer.

The primary antibody was incubated 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 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [RM1323] (AB325777)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling CD66b with ab325777 at 1/20000 (0.025 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human cervical cancer.

The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt

Lab

Flow Cytometry - Anti-CD66b antibody [RM1323] (AB325777)

Flow cytometric analysis of Human neutrophils cells labelling CD66b with ab325777 at 1/5000 (0.01ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD66b antibody [RM1323] (AB325777)

Flow cytometric analysis of Unstimulated HL-60 (Human acute promyelocytic leukemia promyeloblast) (Lower) HL-60 stimulated with 1.25% DMSO for 5 days (Upper) cells labelling CD66b with ab325777 at 1/5000 dilution (0.01ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD66b antibody [RM1323] (AB325777)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HL-60 (human acute promyelocytic leukemia promyeloblast) HL-60 cells treated with DMSO (1.25%) for 5 days cells labelling CD66b with ab325777 at 1/50 (9.94 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased cytoplasmic staining in differentiated HL-60 cells (treatment with DMSO (1.25%) for 5 days), (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-CD66b antibody [RM1323] (AB325777)

CD66b was immunoprecipitated from 0.35 mg HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate with ab325777 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325777 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate
Lane 2 : ab325777 IP in HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325777 in HL-60 whole cell lysate

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

Exposure time : 93 seconds.

All lanes:

Immunoprecipitation - Anti-CD66b antibody [RM1323] (ab325777) at 1/1000 dilution

Lane 1:

HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 10 µg

Lane 2:

ab325777 at 1/30 IP in HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325777 in HL-60 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 95 kDa

false

Exposure time: 93s

• WB

Lab

Western blot - Anti-CD66b antibody [RM1323] (AB325777)

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

Low expression : liver and cerebellum.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD66b antibody [RM1323] (ab325777) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

human spleen tissue lysate at 20 µg

Lane 3:

Human cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 95 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD66b antibody [RM1323] (AB325777)

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

Negative control : SW1463 and HCC827.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD66b antibody [RM1323] (ab325777) at 1/1000 dilution

Lane 1:

SW1463 (human Large intestine Rectum epithelial) whole cell lysate at 20 µg

Lane 2:

HCC827 (human lung adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Untreated HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 4:

HL-60 treated with 1.25% Dimethyl sulfoxide (DMSO) for 5 days whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 95 kDa,36 kDa

true

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [RM1323] (AB325777)

Immunohistochemical analysis of paraffin-embedded (A) : HEK-293T cells transfected with a CEACAM8 expression vector containing a his tag. (B) : HEK-293T cells transfected with a CEACAM1 expression vector containing a his tag. (C) : HEK-293T cells transfected with a CEACAM3 expression vector containing a his tag. (D) : HEK-293T cells transfected with a CEACAM5 expression vector containing a his tag. (E) : HEK-293T cells transfected with a CEACAM6 expression vector containing a his tag. (F) : HEK-293T cells transfected with empty vector containing a his tag. tissue labeling CD66b with ab325777 at 1/20000 (0.025 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on (A) HEK-293T cells transfected with a CEACAM8 expression vector containing a his tag. No staining on HEK-293T cells transfected with a CEACAM1 (B), CEACAM3 (C), CEACAM5 (D), CEACAM6 (E), and empty (F) expression vector containing a his tag.

This antibody does not cross-react with CEACAM1, CEACAM3, CEACAM5 and CEACAM6 proteins.

The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

• WB

Lab

Western blot - Anti-CD66b antibody [RM1323] (AB325777)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);Anti-6X His tag® antibody [AD1.1.10] (ab15149) (27-150KDa).

All lanes:

Western blot - Anti-CD66b antibody [RM1323] (ab325777) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a His-tag, whole cell lysate at 5 µg

Lane 2:

293T cells transfected with a human CEACAM1/CD66a expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 3:

293T cells transfected with a human CEACAM3/CD66d expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 4:

293T cells transfected with a human CEACAM5/CD66e expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 5:

293T cells transfected with a human CEACAM6/CD66c expression vector containing a His-tag, whole cell lysate at 5 µg

Lane 6:

293T cells transfected with a human CEACAM8/CD66b expression vector containing a His-tag, whole cell lysate at 5 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 95 kDa,36 kDa,27-150 kDa

false

Exposure time: 15s