Donkey antimouse igg h l alexa fluor 488

For immunofluorescent double labelling, sections were incubated with 15% normal donkey serum at room temperature for 30 min and then incubated overnight at 4°C with primary antibodies. After washing, sections were incubated with secondary antibodies for 2 h. The primary antibodies were polyclonal goat anti-LCN2 (1:200 dilution, R&D system), polyclonal rabbit anti-SLC22A17 (marker for LCN2 receptor, 1:50 dilution, Abcam), polyclonal mouse anti-GFAP (1:400 dilution, Millipore), monoclonal mouse anti-neuronal nuclei (NeuN; 1:100 dilution, Millipore) and polyclonal goat anti-Iba1 (1:500 dilution, Abcam) or rabbit anti-Iba-1 (1:500 dilution, Wako). The secondary antibodies were donkey antigoat IgG(H+L) Alexa Fluor 594 (1:500 dilution, Invitrogen), donkey antirabbit IgG(H+L) Alexa Fluor 594 (1:500 dilution, Invitrogen), donkey antimouse IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen), donkey antirabbit IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen) and donkey antigoat IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen). The double labelling was analysed using a fluorescence microscope.

Sections were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-Block^TM Histochemical Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and then stained with the following antibodies: polyclonal rabbit anti-calbindin (1:500, CB38, Swant, Switzerland) and monoclonal mouse anti-fetuin-A (diluted 1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Sections were then incubated with donkey anti-mouse IgG (H + L) Alexa Fluor 488 or donkey anti-rabbit IgG (H + L) Alexa Fluor 594 (1:300, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. Fluoroshield^TM with DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used for nuclear staining. Stained brain sections were analyzed with an immunofluorescence microscope (Thermo Fisher Scientific, Invitrogen^TM EVOS^TM M7000 Imaging System, USA). These stained brain sections were quantified with the ImageJ software v1.52a (Bethesda, MD, USA).
Cells were plated onto sterilized glass coverslips placed in 6- or 24-well culture plates. Cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, USA), blocked with CAS-Block^TM Histochemical Reagent (ThermoFisher Scientific, Waltham, MA, USA), and stained as described above.