Porcine trypsin solution

The HT29 and WiDr human colorectal adenocarcinoma cell lines, and the CCD-18Co (ATCC^® CRL-1459^™) human fibroblast cell line isolated from normal colon tissue (both from American Type Culture Collection, Manassas, VA, USA) were cultured in 75 cm² flasks (TPP AG, Trasadingen, Switzerland) in RPMI-1640 (Euroclone, Milano, Italy) supplemented with 10% (v/v) fetal calf serum (FCS) (Euroclone, Milano, Italy), 100 U/mL penicillin G, 40 µg/mL gentamycin sulfate, and 2.5 µg/mL amphotericin B (all from Sigma-Aldrich, Milano, Italy) at 37 °C in a humidified 5% CO₂ atmosphere. Cells were harvested twice a week with 0.25% porcine trypsin solution (Sigma-Aldrich, Milano, Italy).

To determine the presence of Schwann cells more specifically, porcine sciatic branches were immunolabelled for the glial marker S100β. Initially, antigen retrieval of samples was carried out by adding 100 μL of 0.17% trypsin working solution [1% (v/w) calcium chloride (VWR) solution added to 0.5% (v/v) porcine trypsin solution (Sigma)] to each section and incubated at 37 °C for 20 min and left to cool for a further 10 min at room temperature. Samples were then incubated with 7.5% (w/v) BSA diluted in PBS at room temperature for 60 min, followed by washing once with 1% (w/v) BSA in PBS. The nerve tissue samples were incubated with primary rabbit anti-S100β antibody [polyclonal IgG; Abcam, ab868, 1: 50 (v/v) in 1%; (v/v) BSA] at 4 °C overnight, followed by washing three times with PBS for 5 min and then incubated with secondary FITC-conjugated anti-rabbit IgG [Abcam UK, ab97050, 1: 500 (v/v)] at room temperature in the dark for 1 h. Each section was washed three times with PBS for 5 min and counterstained with 300 nm DAPI in PBS and incubated for 20 min in the dark at room temperature. Sections were finally washed three times with PBS for 5 min and imaged using a Zeiss LSM510 META upright/inverted confocal microscope [xenon arc lamp to excite FITC (λ_ex = 495 nm/λ_em = 515 nm)]. Nuclei were visualised using excitation at λ_ex = 510 nm and emission capture at λ_em = 610–650 nm.