Mammary gland tissue from lactating light horse mare was incubated with ab2772 at 1:100 for 1 hour at 23°C and for 24 hours at 4°C. A biotin-conjugated goat anti-mouse IgG was used as the secondary antibody (23°C for 1 hour) .Development using avidin-biotin complex 23°C for 1 hour plus 3,3-diaminobenzadine for 15 min and counterstained with haematoxylin. Antigen retrieval was facilitated using steaming at 95°C for 20 min in Trizma® buffer (pH 10.0) . Blocking endogenous peroxidase performed with 0.3% H2O2 and MeOH (20 min at 23°C). To decrease non-specific staining, tissue was pre-incubated in normal goat serum for 30 min at 23°C.

Immunocytochemistry/Immunofluorescence analysis of Prolactin Receptor/PRL-R shows staining in A2058 cells. Prolactin Receptor/PRL-R (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2772 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Prolactin Receptor/PRL-R shows staining in HepG2 cells. Prolactin Receptor/PRL-R (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2772 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Prolactin Receptor/PRL-R shows staining in MCF-7 cells. Prolactin Receptor/PRL-R (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2772 (1:20) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

ab2772 staining the Prolactin Receptor/PRL-R in Human salivary gland tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 10% serum for 1 hour at 20°C; antigen retrieval was not performed. Samples were incubated with primary antibody (1/100) for 12 hours at 4°C. A Biotin-conjugated Rabbit anti-mouse polyclonal (1/200) was used as the secondary antibody.

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