Recombinant Anti-HDAC3 antibody [Y415] (ab32369)

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma sections labelling HDAC3 with purified ab32369 at dilution of 1:50. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunocytochemistry/Immunofluorescence analysis of K562 cells labelling HDAC3 with purified ab32369 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilized using 0.1% Triton X-100. ab150077, Alexa Fluor^®488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^®594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used and for negative control 2, mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used. 

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling HDAC3 with purified ab32369 at 1/30 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Purified ab32369 at 1/20 immunoprecipitating HDAC3 in K562 whole cell lysate observed at 49 KDa (lanes 1 and 2). 

Lane 1 (input): K562 whole cell lysate 10ug              

Lane 2 (+): ab32369 + K562 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32369 in K562 whole cell lysate 

The secondary antibody used was VeriBlot for IP Detection Reagent (HRP) (ab131366) for detection at dilution of 1/1000.            

Blocking/Diluting buffer 5% NFDM/TBST. 

Paraffin-embedded human ovary carcinoma
ab32369 at 1/250 dilution

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