Spermatogenic phenotypes of Rnf8;Scml2-dKO mice.

Immunostaining of testicular sections with WT1 (ab89901), a marker of Sertoli cells, and PLZF, a marker of undifferentiated spermatogonia.

For preparation of testicular paraffin blocks, testes of mutants and littermate controls were fixed with 4% paraformaldehyde at 4°C overnight. Testes were then dehydrated and embedded in paraffin. For histological analyses, 6 μm-thick paraffin sections were deparaffinized and autoclaved in Target Retrieval Solution at 121°C for 20 min. The sections were blocked with for 10 min at room temperature, and then incubated with primary antibodies at 4°C overnight.

Immunocytochemsitry/Immunofluorescence analysis of K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling Wilms Tumor Protein (green) with purified ab89901 at 1/50.

Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

All lanes : Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2] (ab89901) at 1/500 dilution

Lane 1 : Human testis whole cell lysate. at 15 µg
Lane 2 : K562 (Human T cell leukemia T lymphocyte) whole cell lysates at 4 µg
Lane 3 : Mouse testis whole cell lysates at 15 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa

Exposure time: Left image: 3 minutes
                         Right image: 15 seconds

This antibody shows low affinity in mouse sample.

Blocking and diluting buffer: 5% NFDM/TBST

 

 

Flow cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling Wilms Tumor Protein with ab89901 at 1/200 dilution (0.1 μg)/ Red.

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol.  A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (ab172730) / Black was used as the isotype control. Cells without incubation with primary antibody and secondary antibody / Blue was used as the unlabeled control.

Podocyte counting using WT1 staining.

Mouse kidney sections were stained with WT1 antibody ab89901 using immunohistochemistry. (A) Each glomeruli was identified and numbered using ImagePro premier imaging software. (B) A tracing was applied along the Bowman’s capsule to assess the surface area of the glomerulus. (C) Each WT1 positive nucleus was identified and the nuclear size assessed.

Immunohistochemical analysis of fromaldehyde fixed, paraffin embedded rat testis tissue sections, labeling Wilms Tumor Protein using ab89901.

Heat mediated antigen retrival was performed using 10 mM sodium citrate and 0.05% Tween-20. Tissue sections were incubated with ab89901 at a 1/50 dilution for 12 hours at 4ºC. The tissues were blocked with 10% serum for 30 minutes at 25ºC. The secondary used was a Donkey CY3^® conjugate at a 1/200 dilution.

See Abreview

ab89901 staining Wilms Tumor Protein in paraffin-embedded human ovarian serous adenocarcinoma tissue sections by Immunohistochemistry.

Antigen retrieval was by heat mediation using citrate buffer (pH 6.0) was performed. Samples were incubated with primary antibody at 1:500 dilution (0.49 μg/ml). A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Nuclear staining on human ovarian serous adenocarcinoma (PMID: 11939727) is observed.

ab89901 staining Wilms Tumor Protein in paraffin-embedded mouse testis tissue sections by Immunohistochemistry.

Antigen retrieval was by heat mediation using citrate buffer (pH 6.0). Samples were incubated with primary antibody at 1:500 dilution (0.49 μg/ml). A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Nuclear staining on Sertoli cells in mouse testis (PMID: 21863216) is observed.

ab89901 staining Wilms Tumor Protein in paraffin-embedded human kidney tissue sections by Immunohistochemistry.

Antigen retrieval was by heat mediation using citrate buffer (pH 6.0). Samples were incubated with primary antibody at 1:500 dilution (0.49 μg/ml). A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Nuclear staining on human kidney glomerulus (PMID: 12898605) is observed.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human Wilms tumor tissue labeling Wilms Tumor Protein with unpurified ab89901 at 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human fetal tissue labeling Wilms Tumor Protein with unpurified ab89901 at 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2] (ab89901) at 1/100 dilution (unpurified) + K562 (Human chronic myelogenous leukemia cell line from bone marrow) cell lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2] (ab89901) at 1/5000 dilution

Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast). Whole cell lysates
Lane 2 : THP-1 (Human monocytic leukemia monocyte). Whole cell lysates
Lane 3 : SH-SY5Y (Human neuroblastoma epithelial cell) Whole cell lysates
Lane 4 : HeLa (Human cervix adenocarcinoma epithelial cell) Whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 55 kDa


Exposure time: 3 minutes

WT1 expression varies in different cell lines. 

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2] (ab89901) at 1/1000 dilution (purified) + K562 (Human chronic myelogenous leukemia cell line from bone marrow) cell lysate at 10 µg

Secondary
Peroxidase-conjugated goat anto-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Wilms Tumor Protein antibody [CAN-R9(IHC)-56-2] (ab89901) at 1/1000 dilution (unpurified) + Ramos (Human Burkitt's lymphoma cell line) cell lysate

Predicted band size: 55 kDa