IHC image of ab1801 staining Actin in normal human colon 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 ab1801, 5μl/ml 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 haematoxylin and mounted with DPX. No primary antibody was used in the negative 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

Western blot analysis of HeLa cells treated for 12 hours with hesperidin (h) (2.5 μg/ml, 4,01 μM), mangiferin (5 μg/ml, 11.84 μM) (m), and hesperidin (2.5 μg/ml, 4.01 μM) in a presence of mangiferin (5 μg/ml, 11.84 μM) (h+m). Immunoblotting was performed with the following primary antibodies: Bax (ab32503), BCL2 (ab59348), beta actin (ab1801), and caspase 8. After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H+L) or with goat anti-mouse IgG (H+L) HRP-conjugated secondary antibodies and detected using ECL. Densitometry was performed using Image Lab software v. 4.1 (BioRad).

Top panel: Following 12h of treatment of HeLa cells with hesperidin (h), mangiferin (m), and hesperidin in a presence of mangiferin (h+m), the mRNA levels were monitored in real - time PCR experiments. The BAX and BCL2 mRNA levels results from 2 independent experiments (n?=?8) are plotted relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S rRNA levels and expressed as a fold change over the EtOH control. Error bars represent standard derivations.

Bottom panel: Following 12h of treatment of HeLa cells with hesperidin (h), mangiferin (m), and hesperidin in a presence of mangiferin (h+m), the protein levels of Bax and BCL2 were detected with SDS-PAGE and Western Blot and related to beta actin levels.

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