Anti-PLCZ1 antibody [EPR29768-46]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human testis tissue staining PLCZ1 with ab325528 at a 1/500 (1.028 μg/ml) dilution, ab270534 anti-DDX4 used at a 1/2000 (0.238 μg/ml) dilution, and ab267377 anti-Wilms tumor protein used at a 1/500 (0.706 μg/ml) dilution.

Panel A : anti-PLCZ1 (green; Opal™520), anti-DDX4 (magenta; Opal™690), anti-Wilms tumor protein (gray; Opal™570) on human testis.

Panel B : anti-PLCZ1 staining spermatid in human testis.

Panel C : anti-DDX4 staining spermatogonia 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 ab325528, ab270534 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T cells transfected with a human PLCZ1 expression vector containing a myc-His-tag® (Middle) 293T cells transfected with an empty expression vector containing a myc-His-tag® (Right) cells labelling PLCZ1 with ab325528 at 1/5000 dilution (0.01ug) / Middle and 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling PLCZ1 with ab325528 at 1/500 (1.028 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human testis (PMID : 26721930, PMID : 32146562). 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling PLCZ1 with ab325528 at 1/5000 (0.103 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 positive staining in 293T cells (shown in green) transfected with a human PLCZ1 vector containing a myc-His-tag®. 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).

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/100 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-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded (A) : HEK-293T (human embryonic kidney epithelial cell) transfected with a human PLCZ1 expression vector containing a myc-His-tag (B) : HEK-293T transfected with empty vector containing a His tag tissue labeling PLCZ1 with ab325528 at 1/1000 (0.514 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) : HEK-293T transfected with a human PLCZ1 expression vector containing a myc-His-tag, no staining on (B) HEK-293T transfected with empty vector containing a His tag. 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-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling PLCZ1 with ab325528 at 1/500 (1.028 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no 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-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling PLCZ1 with ab325528 at 1/500 (1.028 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human kidney. 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

• I-ELISA

Lab

Indirect ELISA - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Indirect ELISA analysis of ab325528 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human PLCZ1 protein.

Antigen concentration : 1000 ng/ml

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (perfused fixed) tissue labeling PLCZ1 with ab325528 at 1/200 (2.57 μg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 μg/ml dilution (Green).

Low expression : confocal image showing positive staining on mouse spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with the primary antibody for 60 mins at room temperature. The section was then mounted using Fluoromount™.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 μg/ml dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse testis (perfused fixed) tissue labeling PLCZ1 with ab325528 at 1/200 (2.57 μg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor®488) preadsorbed at 1/1000 2 μg/ml dilution (Green).

Confocal image showing positive staining on mouse testis. The nuclear counterstain was DAPI (Blue). The section was incubated with the primary antibody for 60 mins at room temperature. The section was then mounted using Fluoromount™.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor®488) preadsorbed at 1/1000 2 μg/ml dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling PLCZ1 with ab325528 at 1/1000 (0.514 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse 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 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-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling PLCZ1 with ab325528 at 1/1000 (1.028 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on mouse spleen (PMID : 14697805). 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PLCZ1 antibody [EPR29768-46] (AB325528)

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized mouse sperm; Mouse PBMC (mouse primary peripheral blood mononuclear cell) cells labelling PLCZ1 with ab325528 at 1/50 (10.28 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 mouse sperms (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Negative control : mouse PBMC. 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.