Anti-CDCA2 antibody [EPR28722-6]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling CDCA2 with ab325345 at 1/100 (5.16 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human testis. 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 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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Immunohistochemical analysis of paraffin-embedded Human lymph node tissue labeling CDCA2 with ab325345 at 1/100 (5.16 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human lymph node. 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 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

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human testis tissue staining CDCA2 with ab325345 at a 1/100 (5.16 μg/ml) dilution, ab315240 anti-Perlecan used at a 1/2000 (0.254 μg/ml) dilution, and ab267377 anti-Wilms tumor protein used at a 1/500 (0.706 μg/ml) dilution.

Panel A : anti-CDCA2 (green; Opal™520), anti-Perlecan (magenta; Opal™690), anti-Wilms tumor protein (gray; Opal™570) on human testis.

Panel B : anti-CDCA2 staining mitotic cells in human testis.

Panel C : anti-Perlecan staining basement membrane and endothelium in human testis.

Panel D : anti-Wilms tumor protein staining Sertoli cells in human testis.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325345, ab315240 and ab267377 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human testis tissue staining CDCA2 with ab325345 at a 1/100 (5.16 μg/ml) dilution, ab318972 anti-COL8A1 used at a 1/2000 (0.264 μg/ml) dilution, and ab267377 anti-Wilms tumor protein used at a 1/500 (0.706 μg/ml) dilution.

Panel A : anti-CDCA2 (green; Opal™520), anti-COL8A1 (magenta; Opal™690), anti-Wilms tumor protein (gray; Opal™570) on human testis.

Panel B : anti-CDCA2 staining mitotic cells in human testis.

Panel C : anti-COL8A1 staining basement membrane in human testis.

Panel D : anti-Wilms tumor protein staining Sertoli cells in human testis.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325345, ab318972 and ab267377 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling CDCA2 with ab325345 at 1/50 (10.32 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 nuclear staining in HeLa cells (shown in green). In early mitosis, signal is observed at the periphery of chromosomes (arrow). 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling CDCA2 with ab325345 at 1/100 (5.16 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human ovarian 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 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

• WB

Lab

Western blot - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

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

To minimize protein degradation, lysates were made fresh.The identity of the bands below 75 kDa are unknown.

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

Exposure time : Lane 1 : 125 seconds; Lanes 2-3 : 180 seconds

All lanes:

Western blot - Anti-CDCA2 antibody [EPR28722-6] (ab325345) at 1/1000 dilution

Lane 1:

DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

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

The antibody recognizes a band at the expected size (just above the 97 kDa marker) in HCT116 and HEK293 cells. This band is missing in the HCT116 CDCA2/RepoMan KO cell line, and depleted when CDCA2/RepoMan is knocked down in HEK293 cells by lentiviral-driven shRNA expression. The image was kindly provided by the Trinkle-Mulcahy lab (University of Ottawa).

All lanes:

Western blot - Anti-CDCA2 antibody [EPR28722-6] (ab325345) at 1/1000 dilution

Lane 1:

HCT116 parental at 40 µg

Lanes 2 - 3:

HCT116 CDCA2/RepoMan knockout at 40 µg

Lane 4:

HEK293 untreated at 40 µg

Lane 5:

HEK293 infected with shRNA control (48 hours) at 40 µg

Lane 6:

HEK293 infected with shRNA control (72 hours) at 40 µg

Lane 7:

HEK293 infected with shRNA targeting CDCA2/RepoMan (48 hours) at 40 µg

Lane 8:

HEK293 infected with shRNA targeting CDCA2/RepoMan (72 hours) at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Observed band size: 110-140 kDa

false

• WB

Lab

Western blot - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

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

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-CDCA2 antibody [EPR28722-6] (ab325345) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting CDCA2 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 1/20000 dilution

Observed band size: 140 kDa,36 kDa

true

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CDCA2 with ab325345 at 1/100 (5.16 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on human skeletal muscle. 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 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

• WB

Lab

Western blot - Anti-CDCA2 antibody [EPR28722-6] (AB325345)

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

The antibody recognizes both endogenous RepoMan and tagged RepoMan in U2OS cells. Transiently overexpressed mStayGold-tagged RepoMan runs higher than the endogenous protein (+37kDa). The image was kindly provided by the Trinkle-Mulcahy lab (University of Ottawa).

All lanes:

Western blot - Anti-CDCA2 antibody [EPR28722-6] (ab325345) at 1/1000 dilution

Lane 1:

U2OS untransfected at 50 µg

Lane 2:

U2OS transfected mStay-Gold-CDCA2/RepoMan at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Observed band size: 110-140 kDa

false