Mouse anti parvalbumin antibody

Sections were quenched in 0.3% H₂O₂ in PBS for 25 minutes, then blocked in 5% goat serum in 0.5% Triton-X TBS for 90 min. Primary antibody mouse anti-parvalbumin antibody (Sigma Aldrich) 1:2000 was added for 2 days followed by a biotinylated goat anti-mouse antibody (Sigma Aldrich) 1:500 for 90 min both in TBS with 0.5% Triton-X. Following this, A and B from the standard Vectastain ABC kit (Vector Laboratories) 1:500 in PBS was added for 1 h. Staining was developed using a DAB (3,3′-Diaminobenzidine tetrahydrochloride hydrate; Sigma Aldrich) solution containing 0.05% DAB and 0.015% H₂O₂ in TBS. Sections were rinsed in PBS and mounted onto slides. After air drying, slides were dehydrated in increasing concentrations of alcohol, cleared with Hemo-De and coverslipped with Fisher Chemical Permount™ Mounting Medium.

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.