Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody

Abstract

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERβ) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization

“…In parallel with our study, another research group has recently worked on the improvement of immunohistochemical detection protocols for ERβ in murine tissues [ 63 ]. The respective research group came up with a protocol that methodologically differs from our protocol in some steps, such as the use of HRP-polymer-bound secondary antibodies instead of the avidin-biotin detection system [ 63 ]. In line with our findings, the authors concluded that the monoclonal antibody PPZ0506 directed against amino acids 2–88 of human ERβ is a suitable mean to detect ERβ in murine tissues.…”

“…Interestingly, the authors present an additional step that can be applied to further reduce non-specific background effects. They show that one way to reduce endogenous Igs is to perfuse the living animals in deep anesthesia with saline buffer and fixative before tissue collection [ 63 ]. To perform such technique, an application requiring approval is a mandatory in the European Union according to Directive 2010/63/EU [ 64 ].…”

“…Therefore, when working with females, precise determination of estrus stage is essential and comparing animals at one stage recommended. If possible, tissues from animals harvested in the diestrus phase should be ideally used [ 24 , 32 , 33 , 63 ]. The possibility of using the same antibody (PPZ0506) in different species brings clarity to the controversial studies of the past years and shows precise interspecies differences in localization of ERβ [ 63 ].…”

“…If possible, tissues from animals harvested in the diestrus phase should be ideally used [ 24 , 32 , 33 , 63 ]. The possibility of using the same antibody (PPZ0506) in different species brings clarity to the controversial studies of the past years and shows precise interspecies differences in localization of ERβ [ 63 ]. Finally, the presented data show that hormonal influences can affect immunohistochemical staining results and non-specific background makes it difficult to delineate positive staining.…”

ERβ in Granulosa Cell Tumors and Its Clinical Potential

“…Single-cell RNA-seq and immunohistochemistry with a validated antibody have determined a highly restricted expression pattern with expression primarily in granulosa cells of the ovary ( Fig. 3A and 3B ) and in human testis ( 7 ), but not mouse testis ( 91 ). The few positive cells in human testis have later been identified as spermatids and spermatocytes through single-cell RNA-seq ( 87 ).…”