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.

Keywords: Esr2; PPZ0506; antibody validation; estrogen receptor beta (ERβ); immunohistochemistry; murine tissue; ovary; staining.

Figure 1

Estrogen receptor beta (ERβ) expression in different tissues. Murine tissues were collected and processed for protein or mRNA analysis as described in Material and Methods: (A) ERβ protein expression was analyzed by western blot analysis in ovarian, uterine, testis and liver tissue extracts with monoclonal antibody PPZ0506. Ponceau S stain confirms successful transfer and β-actin re-probing served as an internal loading control. ERβ protein could only be detected in the murine ovary. Asterisks (*) indicate non-specific bands: (B) in line, the 206 bp Esr2 amplicon amplified by RT-PCR in ovarian tissue was absent in all other analyzed tissues. The product of Rn18s (110 bp) served as a control; (C) immunohistochemical staining for ERβ using PPZ0506 in optimized conditions show strong ERβ-positive granulosa cells (Gr). Further cell types indicated are: #, oocyte cell, T, theca cells; and (D) routine HE staining of murine ovary showed the general histology of the tissue. Scale bar 50 µm (400×).

Figure 2

Heat-induced antigen retrieval is mandatory to obtain specific nuclear estrogen receptor beta (ERβ) staining. Murine ovaries (A) or uterine tissue (B) were used to evaluate whether antigen unmasking is required for specific nuclear ERβ staining using PPZ0506 antibody. Some sections were stained without antigen retrieval, whereas in others antigen retrieval was performed by heating the sections in sodium citrate buffer (10 mM, 0.05% Tween 20, pH 6.0) in a steamer for 30 min. In the depicted experiment, PPZ0506 antibody was diluted 1:6000. Please note that slides without antigen retrieval showed no nuclear staining at all whereas unspecific cytoplasmatic staining occurs (arrowheads). The used antigen retrieval method enabled a dependable and specific nuclear ERβ staining (of the granulosa cells) in the ovary, but no specific staining in the uterus. Scale bars, 100 µm (in all magnifications).

Figure 3

Female reproductive tissues of Esr2-depleted mice are negative for estrogen receptor beta (ERβ) expression. Ovaries (A) and uterine tissue (B) of Esr2 knockout (Esr2^−/−) and wild-type (Esr2^+/+) animals from the same mouse line were stained with PPZ0506 antibody or with isotype-specific IgG_2b negative control. Only granulosa cells (Gr) in Esr2^+/+ ovaries strongly express ERβ, whereas in Esr2^−/− ovary and uterine tissue no specific nuclear staining was observed. Specific staining is shown by arrowheads in Esr2^+/+ and # indicates diffuse non-specific background staining. Scale bar 250 µm (100×) or 50 µm (400×).

Figure 4

Titration of PPZ0506 primary antibody. Paraffin-embedded sections were chosen for immunohistochemical detection of nuclear expressed estrogen receptor beta (ERβ). The antibody (PPZ0506; 1 mg/mL) was diluted to obtain optimal dilution ratio (as indicated) in ovaries (A) or uterus (B). For negative control, IgG isotope staining was performed with a concentration equivalent to primary antibody dilution (1:8000). Cell types of the ovary are indicated as: Gr, granulosa cells; T (arrowheads), theca cells; #, oocyte cells. Please note that the murine uterus tissue was negative for ERβ. Scale bar 50 µm (400×).

Figure 5

Non-specific background signal in uterine tissue. Tissue slices were prepared as described in Material and Methods for routine HE stains or immunohistochemical detection of estrogen receptor beta (ERβ) using PPZ0506 or IgG_2b isotope control. Uterine tissue form mice in (A) the proestrus and (B) the metestrus stage of the estrous cycle are shown. Please note that there are strong differences in non-specific background staining intensity between the different stages. Scale bar 250 µm (100×) or 50 µm (400×).

Figure 6

Effects of VisUBlock in immunohistochemical detection. Tissue slices of murine uterine tissue were prepared as described in Material and Methods including incubation for 1 hour with VisUBlock blocking reagent prior staining with PPZ0506 antibody or isotype-specific IgG_2b negative control. The use of VisUBlock increased unspecific background signals in the uterine tissue. Scale bar: 100 µm (200×).