Ds fi camera

This was performed using a standard immunoperoxidase approach [21 (link)]. Bright-field image acquisition was performed using a Nikon 50i Microscope fitted with a DS-Fi camera and a DSL2 camera control unit (Nikon, Kingston Upon Thames, UK). Antibody details and conditions are described in ESM Table 2.

Eclipse E400 Nikon microscope, with DS-FI camera driven by NIS-D element software (Nikon) was used to capture images (100× magnification) and take the measurements for architectural evaluation of the digestive mucosa. A total of about 30 well-oriented villi and crypts per loop were selected on each section. A villus was measured from the tip to the shoulder (crypt-villus junction) and a crypt was measured from the shoulder to its base (Figure 1c). The crypt-depth to villus-height ratio was calculated to assess the intestinal architectural changes.

DNA fragmentation in apoptotic cells was detected by marking the free DNA end with biotinylated dUTPs (TUNEL) using the in situ Cell Death Detection kit (Ref.11684817910; Sigma-Aldrich) and following the manufacturer’s instructions. Briefly, coronal brain sections containing SNpc were fixed with 4% paraformaldehyde for 20 min, and 3% H₂O₂ was used for endogenous peroxidase inactivation. Tissue was later permeabilized with 0.1% triton and 0.1% sodium citrate for 2 min at 4 °C, and free DNA ends were marked with biotinylated dUTPs in TdT buffer for 60 min. After that, sections were incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min at 37 °C. DNA breaks were observed using a 3-3′-diaminobendicina (DAB) chromogen at a final concentration of 50 μg/mL and H₂O₂. Brain sections treated with 2U of DNAse (Promega, Promega Biotech Ibérica, Alcobendas, Madrid, Spain, Ref.M610A) were used as positive controls for DNA fragmentation. Pictures were taken with a Nikon Eclipse 80i microscope coupled with a Nikon DS-Fi camera.