Western blot of mSin3A on K562 cell extract using ab3479.

Paraffin-embedded human ovary carcinoma tissue (right panel) stained for mSin3A using ab3479 at 1/200 dilution compared to a negative control without primary antibody (left panel) in immunohistochemical analysis, followed by HRP-conjugated secondary antibody. Detection: DAB staining.

Antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min.

ChIP analysis of Sin3A was performed using cross-linked chromatin from 1x10⁶ HCT 116 (human colorectal carcinoma cell line) cells treated with serum for 0, 15, and 30 minutes. IP was performed using a multiplex microplate Matrix ChIP assay with 1.0 µl/100 µl well volume of ab3479. Chromatin aliquots from ~1x10⁵ cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µl of eluted DNA in 2 µl SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 or exon-2 of Egr1. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. 

Immunocytochemical immunoflurescence analysis of NIH-3T3 cells labelling mSin3A using ab3479. Formalin-fixed cells were permeablized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were then incubated with ab3479 in 3% BSA-PBS at a dilution of 1:100 overnight in a 4°C high humidity environment. Cells were then washed with PBST and incubated with a green DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Cells were counterstained with DAPI or Hoechst labelling the nuclear DNA blue. The Left Image is a negative control without the prescence of ab3479. Image magnification is 60X.

Immunocytochemical immunoflurescence analysis of HeLa cells labelling mSin3A using ab3479. Formalin-fixed cells were permeablized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were then incubated with ab3479 in 3% BSA-PBS at a dilution of 1:200 overnight in a 4°C high humidity environment. Cells were then washed with PBST and incubated with a green DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Cells were counterstained blue with DAPI or Hoechst labelling the nuclear DNA and red against actin using an Alexa Fluor® 554 conjugate. The Left Image is a negative control without the prescence of ab3479. Image magnification is 60X.

Immunocytochemical immunoflurescence analysis of NIH-3T3 cells labelling mSin3A using ab3479. Formalin-fixed cells were permeablized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were then incubated with ab3479 in 3% BSA-PBS at a dilution of 1:100 overnight in a 4°C high humidity environment. Cells were then washed with PBST and incubated with a green DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Cells were counterstained blue with DAPI or Hoechst labelling the nuclear DNA and red against actin using an Alexa Fluor® 554 conjugate. The Left Image is a negative control without the prescence of ab3479. Image magnification is 60X.

ab3479 (2µg/ml) staining mSin3A in human duodenum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclei of epithelial cells.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1/ EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.