Recombinant Anti-HDAC2 antibody [Y461] (ab32117)

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

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

Immunohistochemical analysis of paraffin-embedded fixed (A) Wild-type HEK293T (human embryonic kidney epithelial cell) cell pellet. (B) HDAC2 knockout HEK293T (ab266590) cell pellet staining HDAC2 with ab32117 at 1/10000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Counter-staining used was hematoxylin.
Positive staining on (A) Wild-type HEK293T cell pellet, no staining on HDAC2 knockout HEK293T (ab266590) cell pellet.
The section was incubated with ab32117 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunocytochemistry/ Immunofluorescence analysis of Wild-type HEK293T/HDAC2 KO HEK293T (HDAC2 knockout human embryonic kidney epithelial cell) (ab266590) cells labeling HDAC2 with ab32117 at 1/200 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody at 1/1000 dilution (2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (2.5 µg/ml). DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Confocal image showing nuclear staining in Parental HEK293T cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2 x 10^5 K-562 (Human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab32117 [Y461] . The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

Additional screenshots of mapped reads can be downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

Purified ab32117 at 1:20 dilution (0.5µg) immunoprecipitating HDAC2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg.
Lane 2 (+): ab32117 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32127 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP)(ab131366) (1:5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: kDa

Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labelling HDAC2 with Purified ab32117 at 1:20 dilution (5 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HDAC2 with Purified ab32117 at 1:50 dilution (2.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat testis tissue sections labeling HDAC2 with Purified ab32117 at 1:1500 dilution (0.071 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue sections labeling HDAC2 with Purified ab32117 at 1:1500 dilution (0.071 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling HDAC2 with Purified ab32117 at 1:1500 dilution (0.071 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue sections labeling HDAC2 with Purified ab32117 at 1:1500 dilution (0.071 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. 

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: HDAC2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: A431 whole cell lysate (20 µg)
Lane 4: Hela whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32117 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32117 was shown to specifically react with HDAC2 when HDAC2 knockout samples were used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  ab32117 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 and 1/10000 respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

ab32117 staining HDAC2 in wild-type HAP1 cells (top panel) and HDAC2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32117 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).