Axiocam cm1

Light microscopy photographs and fluorescence microscopy photographs were taken and analyzed exactly as previously reported by^{27 (link)} using a Zeiss Axiostart 50 Fluorescence Microscope equipped with a Zeiss AxioCam Cm1 and (Zeiss Whlk-Contact-Linsfluoreen, Gmb Schconkirchen, Germany) and Floyd Cells Imaging Station microscope. Mean fluorescence intensity (MFI) was obtained by normalizing total fluorescence to the number of nuclei.

Fluorescence microscopy (Zeiss Axiolab A1, Axiocam Cm1) was used to capture images of neurites from five pre‐determined fields of each gel or coverslip using a ×20 lens. The neurite number in the determined fields depended on the cell type and model used (~1–12 neurites). Briefly, the positions of the pre‐determined fields on coverslips were equally spaced (625‐μm apart) in a line across the diameter at the center of the coverslip, for the gels the same method was used but the line of fields was along the edge of the construct where alignment was greatest. The length of each neurite in each field was measured using ImageJ. Following stabilization the gel acquired a thickness of 100 μm and the neurons extended predominantly in a single horizontal plane along the top surface, following the aligned Schwann cells (Phillips, 2014). This allowed analysis of neurite growth to be comparable between both the monolayer and 3D co‐cultures.
Tile scans were used to capture high‐magnification (x20) micrographs from the entire nerve cross‐section using a Zeiss LSM 710 Confocal microscope and images were analyzed using Volocity™ 6.4 (PerkinElmer) running automated image analysis protocols to determine the number of neurofilament‐immunoreactive neurites in each transverse nerve section.

Fluorescence microscopy (Zeiss Axiolab A1, Axiocam Cm1, Carl Zeiss GmbH) was used to capture images of neurites from five pre-determined fields of each gel using a ×20 lens. The positions of the pre-determined fields on the gels were equally spaced (625 μm apart) in a line along the edge of the construct where alignment was greatest [27 (link)]. The length of each neurite in each field (~1–12 neurites) was measured using Fiji ImageJ [29 (link)]. Following stabilization, the gel acquired a thickness of 100 μm and the neurons extended predominantly in a single horizontal plane along the top surface, following the aligned Schwann cells [30 (link)].
Tile scans were used to capture high-magnification (×20) micrographs from the entire nerve cross-section using a Zeiss LSM 710 confocal microscope and images were analysed using Volocity™ 6.4 (PerkinElmer, Beaconsfield, UK) running automated image analysis protocols to determine the number of neurofilament-immunoreactive neurites in each transverse nerve section.

The analysis of markers related to AD, oxidative stress, and cell death was performed exactly as described in ref. [32 (link)]. Briefly, cells subjected to varying treatments were fixed for 20 min with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% BSA. The cells were incubated overnight with primary antibodies described in Table 3. After thorough rinsing, the cells were incubated with secondary fluorescent antibodies, and the nuclei were stained with 1 μM Hoechst 33342 (Life Technologies, Cat# H3570, Carlsbad, CA, USA). Images were acquired on a Zeiss Axio Vert.A1 equipped with a Zeiss AxioCam Cm1.