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Recombinant fragment (Human) corresponding to the external domain
This antibody works well when used in combination with ab972 - Antigen retrieval solution - heat mediated high pH 9.5.
We have found that for IHC application it is very important to use a peroxidase blocking step after the primary antibody incubation to prevent diminished staining. Please see image legend or application note for more details.
Our Abpromise guarantee covers the use of ab846 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration.|
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. The Tris based buffer pH 9.5 is recommended. This antibody may be diluted to a titer of 1/25 - 1/50 in an ABC method. We suggest an incubation period of 1-2 hours at room temperature. However, depending upon the fixation conditions and the staining system employed, optimal incubation and dilutions should be determined by the user. Use peroxidase block after the primary antibody or staining will be diminished!|
|IHC-Fr||Use at an assay dependent concentration. Use peroxidase block after the primary antibody or staining will be diminished. Post-fixation with 10% formalin after the primary antibody may improve antibody staining.|
ab846 staining CD4 in Human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). The tissue was paraformaldehyde fixed and then an antigen retieval step was performed (heat mediated) prior to incubating the cells with the antibody for 15 minutes. Samples were incubated with primary antibody (1/50) for 30 minutes at RT.
IHC image of CD4 staining in a section of formalin-fixed paraffin-embedded Human normal spleen*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab846 at 1/50 dilution. After incubation with ab846, hydrogen peroxide (1.6%, for 30min at room temperature) was used to block the tissue section. Following peroxidase blocking a HRP-conjugated secondary (Ab97040, 1/500 dilution) was used for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/50 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
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