Biotinylated goat anti rabbit igg and streptavidin peroxidase complex

Paraffin sections were processed for deparaffinization with xylene and for rehydration with ethanol, and treated with an antigen-resuscitation solution (Dako, Santa Clara, CA, USA) in an autoclave for 15 min at 121 °C. After washing with PBS, sections were immersed in protein block, serum-free (Dako), for 15 min. Sections were incubated with diluted anti-glucocorticoid receptor (CST, Danvers, MA, USA, Table 1) and anti-β2AR (Abcam, Cambridge, UK, Table 1) at 4 °C overnight. Subsequently, sections were treated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Nichirei Biosciences Inc., Tokyo, Japan) for 30 min. After staining with diaminobenzidine and counterstaining with hematoxylin, sections were observed and captured under a light microscope (VS120, Olympus Corporation) and a digital camera. To determine the percentage of GR- and β2AR-positive cells in the metastatic nodules, 5 sections per animal (n = 5) and 5 fields per section were randomly selected for evaluation. All measurements were conducted in a double-blind procedure, as previously reported [24 (link)].

Paraffin sections of the mouse ovaries and uterus were deparaffinized in xylene and rehydrated in an ascending series of ethanol solution. After antigen retrieval, sections were treated with blocking solution. Sections were incubated with anti-β-catenin (1:1000, Santa Cruz, Dallas, TX, USA), anti-cytochrome P450 family 19 subfamily a member 1 (Cyp19a1, 1:100, Assay Biotechnology Company, Inc., Sunnyvale, CA, USA), and anti-Wnt10a (1:5000). For generating anti-Wnt10a antibody, rabbits were immunized with a synthetic peptide corresponding to amino acids 160-172 of the mouse Wnt10a. The sera of the immunized rabbit were then isolated and purified 19 (link). Sections were then incubated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Nichirei Biosciences Inc., Tokyo, Japan), stained with diaminobenzidine and then counterstained with hematoxylin. Images were captured with a light microscope (Olympus VS120) and digitized with software VS-ASW (Olympus).

Brain tissues were fixed in 4% neutral buffered paraformaldehyde (pH = 7.4) and embedded in paraffin. The paraffin sections (5 μm) were deparaffinized in xylene and rehydrated in ethanol; then, antigen retrieval was performed by incubating in oiled 0.01 M sodium citrate buffer (pH = 6) and blocking endogenous peroxidase activity using 10% H2O2. To reduce nonspecific staining, the slides were immersed in Protein Block, Serum Free (Dako, Tokyo, Japan) for 15 min. For immunohistochemical staining, the sections (5 μm) were incubated with anti-BDNF (1:100, Abcam, Cambridge, UK), anti-GR (1:400, Cell Signaling Technology, Danvers, MA, USA) or anti-doublecortin (1:1000, Abcam, Cambridge, UK) and then incubated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature, stained with diaminobenzidine and then counterstained with hematoxylin. A light microscope (Olympus, BX50, Tokyo, Japan) connected to a digital camera was used for examining and photographing the slides. For quantification of doublecortin-positive cells in the DG of both hippocampal lobes, the number of immunopositive cells was counted in 10 randomly selected fields of sections, original magnification ×400, as previously described [72 (link)].