Dfc320 r2 digital camera

The fixed testes were dehydrated in increasing ethanol concentrations before embedding in paraffin. Then, 5-µm-thick paraffin sections were stained with hematoxylin (#MHS1; Sigma Aldrich; Milan, Italy) and eosin (HT110216; Sigma Aldrich; Milan, Italy) for histological evaluation. Slides were examined with a Leica microscope (Leica DM 2500, Leica Microsystems, Wetzlar, Germany). Photographs were taken using Leica DFC320 R2 Digital Camera. For histopathological analysis, 30 seminiferous tubules/animal, for a total of 150 tubules per group, were counted.

To determine EHBP1L1 localization in rat and human SPZ, the samples were first fixed in 4% paraformaldehyde in PBS for 10 min and then washed twice in PBS. The slides were incubated with 0.1% (v/v) Triton X-100 in PBS for 30 min. Later, nonspecific binding sites were blocked with 5% BSA and normal goat serum diluted 1:5 in PBS before the addition of the anti-EHBP1L1 primary antibody, as described above, and overnight incubation at 4 °C. After three washes in PBS, slides were incubated for 1 hr with the secondary antibody (#A32731; Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in the blocking mixture and with PNA lectin diluted 1:50. The slides were washed again in PBS and then mounted with Vectashield + DAPI (Vector Laboratories, Peterborought, UK) for nuclear staining and were then observed under an optical microscope (Leica DM 5000 B + CTR 5000; Leica Microsystems, Wetzlar, Germany) with UV Lamp; images were viewed and saved with IM 1000 software. Photographs were taken using the Leica DFC320 R2 digital camera.

For immunolocalization analysis, 5-µm testis sections were dewaxed, rehydrated, and processed as described by Venditti et al. (48 (link)). Supplementary Table S1 reports the details about all the antibodies used. The slides were mounted with Vectashield + DAPI (#H-1200–10; Vector Laboratories, Peterborough, UK) for nuclear staining and then observed under an optical microscope (Leica DM 5000 B + CTR 5000; Leica Microsystems, Wetzlar, Germany) with a UV lamp. The images were analyzed and saved with IM 1000 software (version 4.7.0; Leica Microsystems, Wetzlar, Germany). Photographs were taken using the Leica DFC320 R2 digital camera. Densitometric analysis of immunofluorescence (IF) signal intensity and counting of positive cells was performed with the Fiji plugin (version 3.9.0/1.53 t) of ImageJ Software counting 30 seminiferous tubules/animal for a total of 150 tubules per group. Each IF was performed in triplicate.