Axio scope optical microscope

Optical microscopy was performed using a Zeiss Axio.Scope optical microscope in Köhler illumination equipped with a 100× objective (Zeiss EC Epiplan-APOCHROMAT 0.95 HD DIC) coupled to a spectrometer (Avantes HS2048) via an optical fibre (Thorlabs, FC-UV50-2-SR). Five spectra were collected for each sample using an integration time of 10 ms and 20 ms for reflection and transmission measurements, respectively. The reflectance and transmittance were calculated using a silver mirror (ThorLabs, PF10-03-P01) and the glass substrate as references respectively.

Both eyes of all mice were fixed in Davidson’s solution for 2 days and then embedded in paraffin. Paraffin-embedded tissues were sectioned at a thickness of 3 μm. H&E staining was performed according to a standard protocol. Sections between 100 and 200 μm from the optic nerve of both eyes were observed at 400× magnification using an Axio Scope optical microscope (ZEISS, Oberkochen, Germany). The thicknesses of the whole retina, photoreceptor segment layer (PSL), inner nuclear layer (INL), and outer nuclear layer (ONL) were measured using the Axio Vision SE64 (ZEISS) program. The number of nuclei in the ONL was analyzed using the Image J software [17 (link)]. For immunohistochemistry (IHC), sectioned tissue was stained with an anti-Superoxide dismutase 1 (SOD1) antibody (ab51254, Abcam, Cambridge, UK). Images were acquired using a confocal laser scanning microscope (Nikon, Minato-ku, Japan) and quantified using the Image J software [18 (link)].