Optimized selection of specific antibodies against rodent ESR1 proteins and their application for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506.

The development of immunohistochemical and immunohistofluorescence assays is essential for investigating the tissue and cellular distribution of target proteins. In this study, we identify specific anti-ESR1 antibodies against rodent ESR1 proteins and evaluate their applicability for immunohistochemistry and dual immunohistofluorescence with the specific anti-ESR2 antibody PPZ0506. We assessed the specificity and cross-reactivity of six commercially available anti-ESR1 antibodies (Clones MC-20, C1355, E115, H4624, SP1, and F-10) against mouse and rat ESR1 proteins using immunoblotting and immunocytofluorescence assays. Among them, MC-20, C1355, E115, and H4624 exhibited specific immunoreactivity to mouse and rat ESR1 proteins. These four antibodies were subsequently applied to paraffin-embedded ovarian and uterine sections from mice and rats. Heat-induced antigen retrieval and an appropriate antibody dilution were required to obtain specific and adequate signals. MC-20 and E115 were suitable for immunohistochemical detection of ESR1 proteins, while C1355 was effective for uterine tissue staining. H4624 showed utility only in mouse tissues. Furthermore, rabbit-derived MC-20 and E115 antibodies were successfully employed in dual immunohistofluorescence assays with the mouse monoclonal PPZ0506 antibody, enabling simultaneous visualization of ESR1 and ESR2 proteins in paraffin-embedded ovarian sections. Notably, little cellular co-localization of ESR1 and ESR2 proteins was observed in mouse and rat ovarian sections. These findings provide a validated set of antibodies for ESR1 immunohistochemical detection and demonstrate their compatibility with ESR2 co-labeling, facilitating detailed analysis of estrogen receptor distribution in rodent tissues.