Bm4000 b led microscope

Four non-serial glandular sections per animal were used, and the analyses were performed using a BM4000 B LED microscope (Leica) and the Leica Application Suite software LAS 4.3 (Leica Microsystems, Wetzlar, Germany). For each marker, threshold adjustment and colour range (hue, saturation and intensity) were rigorously standardized in all sections during the analyses.

The analysis of the immunofluorescence was performed using a DFC 550 Camera (Leica) attached to a BM4000 B LED microscope (Leica), and the Leica Application Suite software (LAS 4.3, Leica) .
The measurement of AR, KPNA and SHBG immunoexpression was performed in the proximal region of four non-serial sections of the cauda epididymidis per animal. In the AR and KPNA immunolabeled sections, a standardized total epithelial area of 50,000 µm 2 was measured. In this area, the nuclear and cytoplasmic AR immunofluorescent areas as well as KPNA immunofluorescent area were measured, and the area per µm 2 of epithelium was obtained. The AR immunofluorescent nuclear/ cytoplasmic ratio was also calculated. SHBG immunofluorescent area was measured in a standardized total connective tissue area of 560,000 µm 2 per animal, and the immunofluorescent area/µm 2 of connective tissue was calculated.
During each immunoreaction analyzed in the sections from CG and CMTG, all the parameters of the software, including threshold adjustment and color range -hue, saturation and intensity (LAS 4.3, Leica) -were rigorously calibrated/ standardized for each image analyzed.