Anti-CD66b antibody [EPR25354-2]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [EPR25354-2] (AB300122)

Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection) was used.

Positive staining on neutrophils in human stomach carcinoma (PMID : 31329780).

The section was incubated with ab300122 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD66b antibody [EPR25354-2] (AB300122)

Immunohistochemical analysis of paraffin-embedded human stomach tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection) was used.

Positive staining on neutrophils in human stomach (PMID : 32096331).

The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300122) followed by ready to use secondary antibody LeicaDS9800 (BOND^™ Polymer Refine Detection).

Heat mediated antigen retrieval 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 [EPR25354-2] (AB300122)

Immunohistochemical analysis of paraffin-embedded : (A) HEK-293T cells transfected with a CD66b 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.

All tissues labelling CD66b with ab300122 at 1/20000 (0.027 µg/ml) followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection). This antibody does not cross-react with CEACAM1, CEACAM3, CEACAM5 and CEACAM6 proteins.

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.

The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300150) followed by ready to use secondary antibody LeicaDS9800 (BOND^™ Polymer Refine Detection).

Heat mediated antigen retrieval 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 [EPR25354-2] (AB300122)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection).

Positive staining on human tonsil.

The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300122) followed by ready to use secondary antibody LeicaDS9800 (BOND^™ Polymer Refine Detection).

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

• IP

Lab

Immunoprecipitation - Anti-CD66b antibody [EPR25354-2] (AB300122)

CD66b was immunoprecipitated from 0.35 mg Human spleen tissue lysate 10 µg with ab300122 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300122 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CD66b antibody [EPR25354-2] (ab300122) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 10 µg

Lane 2:

ab300122 at 1/30 IP in human spleen tissue lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300122 in Human spleen tissue lysate at 10 µg

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: 7.75s

• WB

Lab

Western blot - Anti-CD66b antibody [EPR25354-2] (AB300122)

CD66b is a highly glycosylated protein, the molecular weight observed is consistent with what has been described in the literature (PMID : 15147383)

Samples are non-boiled as boiling may cause protein aggregates.

Bis-Tris gel was used for this blot.

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

All lanes:

Western blot - Anti-CD66b antibody [EPR25354-2] (ab300122) at 1/1000 dilution

All lanes:

Human spleen 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

false

Exposure time: 10s