Multimodal Imaging of Oxidative Stress and Calcium Channels in Neurons

We prepared brain sections and performed parvalbumin (PV)/Wisteria floribunda agglutinin (WFA)/8-hydroxy-2′-deoxyguanosine (8-oxo-dG) triple immunofluorescence on males as described in [18 (link)] and Supplemental information. Primary antibodies were mouse monoclonal anti-8-oxo-dG (AMS Biotechnology, Switzerland), rabbit polyclonal anti-PV (Swant, Switzerland), biotin-conjugated lectin WFA (Sigma-Aldrich, Switzerland), while secondary antibodies were goat anti-mouse AF488 (Life Technologies, USA), goat anti-rabbit IgG CY3 (Chemicon International, USA) and streptavidin 405 conjugate (Millipore Corporation, USA).
We carried out CaV3.2/PV and CaV3.3/PV immunohistology as indicated in Supplemental information using the following primary [rabbit anti-CaV3.2 (Santa Cruz Biotechnology, USA); rabbit anti-CaV3.3 (Alomone labs, Israel), sheep anti-PV (R&D systems)] and secondary [goat anti-rabbit AF488 and donkey anti-sheep AF594 (Abcam, UK)] antibodies. Confocal image acquisition, and quantification are described in Supplemental information.