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

Abstract

The estrogen receptor beta (ERβ) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERβ, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERβ (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERβ. Herein, we established an immunohistochemical detection protocol for ERβ protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERβ. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERβ. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERβ, is also suitable for reliable detection of murine ERβ. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERβ in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Ovarian ERβ cistrome and transcriptome reveal chromatin interaction with LRH-1

“…ERβ is known to be expressed in granulosa cells, but its expression in other cells including theca cells has been debated [ 19 ]. We performed immunohistochemistry (IHC) with an ERβ antibody (PPZ0506) that has been thoroughly validated across different tissues in humans and rodents [ 17 , 19 – 21 ]. We confirmed that ERβ was strongly expressed in the nucleus of granulosa cells.…”

“…Mice lacking ERα are also infertile and develop cystic follicles [ 15 , 16 ]. ERβ, which is a homolog of ERα, has a noticeably more restricted expression pattern and is predominantly expressed in the granulosa cells of the ovary [ 17 – 21 ]. Its function is also less understood.…”

“…An obstacle for ERβ research has been a lack of specific antibodies [ 17 ]. However, in recent years, a validated, specific antibody has become available [ 17 , 19 , 21 , 32 – 34 ]. In our study, this antibody was used to provide new insight into the role of ERβ in normal ovarian function and fertility.…”

Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging

“…However, their expression differs between functional compartments of the ovary. While estrogen receptor beta (ERb, Esr2) is strongly expressed by granulosa cells in growing follicles (4)(5)(6)(7), estrogen receptor alpha (ERa, Esr1) is more diffusely expressed in interstitial and thecal cells (6)(7)(8). It is therefore not surprising that misregulation or depletion of these receptors has severe effects on physiological processes and impairs fertility (9).…”

Divergent features of ERβ isoforms in triple negative breast cancer: progress and implications for further research

“…The functions of ERβ isoforms may diverge given differences in their three-dimensional structures and abilities to bind to ligands and other molecules. In addition, the function of ERβ may differ among humans, mice, and rats because different lengths and ligand binging affinities have been observed for these orthologs ( Petersen et al, 1998 ; O’Brien et al, 1999 ; Iwamoto et al, 2003 ; Donoghue et al, 2017 ; Schröder et al, 2022 ) ( Figure 1 ). The full-length wild-type ERβ1 isoform is typically referred to as ERβ, unless otherwise stated.…”