Immunohistochemistry was performed on human head and neck squamous cell carcinoma tissue using a rabbit monoclonal antibody against the CCND1 protein ab134175 on 3-µm slides using 224 paraffin sections via the standard SP method.

Panel C: An example of low Cyclin D1 expression.

Panel G: An example of high Cyclin D1 expression.

For full image please see paper.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemical analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, labeling Cyclin D1 with ab134175 at a dilution of 1/200. Cells were paraformaldehyde fixed and permeabilized with 0.5% Triton X-100 in PBS. Incubation with the primary antibody was for 1 hour at 22°C. Cells were counterstained with DAPI following immunostaining.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

IHC image of ab134175 staining Cyclin D1 in rat esophagus formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab134175, 5μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

IHC image of ab134175 staining Cyclin D1 in human esophagus formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab134175, 5μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

ab134175 (purified) at 1/30 immunoprecipitating cyclin D1 in A431 (Human epidermoid carcinoma cell line) cells. For western blotting, an HRP-conjugated goat anti-rabbit IgG, was used as the secondary antibody (1/1000).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Immunofluorescent staining of Ramos (Human Burkitt's lymphoma cell line) cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134175 at a dilution of 1/50. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counterstained with DAPI.

The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Immunohistochemical staining of paraffin embedded human endometrial adenocarcinoma with purified ab134175 at a dilution of 1/100. An HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500 and the sample was counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Unpurified ab134175 staining Cyclin D1 in MCF7 (Human breast adenocarcinoma cell line) cells treated with KN-93 (ab120980).

The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab134175 at 10μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an Goat anti-Rabbit Alexa 488 secondary (ab150081) at 2 μg/ml (shown in green) and Goat anti-Mouse Alexa 594 secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Immunohistochemical analysis of paraffin-embedded human mantle cell lymphoma tissue, labeling Cyclin D1 with unpurified ab134175 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134175).