Axioimage m2 microscope

Tissue sections labeled by immunohistochemistry were imaged for axon reconstructions (see below) or by light microscopy using a Zeiss Axioimage M2 microscope with a Hamamatsu ORCA Flash4.OLT digital camera attached to a HP Z840 PC running Zen 2.3 (blue edition) software. Images of fluorescent immunolabeled sections were acquired using a Keyence BZ-9000 epifluorescent microscope and a Nikon 20×/0.75 Plan-Apo objective attached to a Dell precision T3610 PC running BZ-II Viewer software (v.2.1). For presentation, image files were imported into Adobe Photoshop (Adobe Systems) where linear adjustments to contrast and brightness and cropping was performed. Final figures were composed using Adobe Illustrator (Adobe Systems). Within a figure, all image panels were treated equally unless otherwise noted.

Sections were rinsed with PBS 1X then incubated with blocking solution (8% Goat serum, 0.3% Bovine Serum Albumin, 0.3% Triton, PBS-0.02% Thimérosal) overnight at 4 °C in primary antibody: rabbit anti-FUS antibody (ProteinTech, 11570-1-AP, 1:100) and mouse anti-parvalbumin antibody (Sigma, P3088, 1:1000). After three rinses in PBS, sections were incubated for 2 h at room temperature with Hoechst (Sigma, B2261, 1/50.000) and secondary antibody: Goat anti-mouse Alexa-488 secondary antibody (Invitrogen, A11034, 1:500) and goat anti-mouse Alexa-647 secondary antibody (Invitrogen, A21245, 1:500). Finally sections were subsequently washed with PBS (3 × 10 min) and mounted in Aqua/polymount (Polysciences, 18606).
Images were acquired along the Z axis (Z stacking) using a Zeiss AxioImage.M2 microscope equipped with a Plan-Apochromat ×20/0.8 objective, high performance B/W camera (Orca Flash4, Hamamatsu) and run by the Zeiss Zen2 software. Images were quantified using the ImageJ freeware. First, the user defined ROIs corresponding to the cytoplasm and nucleus or several PV positive cells at several Z positions. Then a homemade macro was used to calculate the ratio, in the green channel, of the cytoplasm intensity divided by the nucleus one.