Figure 4.

Representative micrographs of immunofluorescently (IFC) stained anterior pituitary sections of orchidectomized (Orx) and estradiol-treated orchidectomized (Orx+E) middle-aged rats. Weaker immunofluorescent signal of S100β, stronger growth hormone (GH; adopted from our previous publication [99] and reprinted by permission of the Licensors - publishers Springer (Licence number 4774320912101)) and adrenocorticotropic hormone (ACTH) immunofluorescent signal, and unchanged immunofluorescent signal of subunit β of thyroid-stimulating hormone (TSHβ; adopted from our previous publication [55] and reprinted by permission of the Licensors - publishers Elsevier (Licence number 4774000297301)), respectively, are observable after estradiol treatment. All figures were obtained according to the same procedure described in our earlier papers [55,99]. Briefly, for IFC staining, the following primary antibodies were applied overnight at 4 °C: mouse monoclonal anti-S100β antibody (Abcam, Cambridge, UK; 1:100), polyclonal rabbit anti–rat TSHβ, GH and ACTH (donation from Dr. A. F. Parlow, National Institute of Health, Bethesda, MD, USA; dilutions 1:500, 1:200, 1:200, respectively). Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen Life technologies, CA, USA; 1:300) was applied as secondary antiserum for 1 h. The sections were mounted with Mowiol 4–88 (Sigma-Aldrich, St. Louis, MO, USA). Scale bar is shown in the right corner.