Apochromat na 1.3 oil objective lens

N2a were cultured on coverslips precoated with 0.01 mg/ml poly-L-Lysine (Biochrom) and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). The cells were then washed in PBS and incubated for 1 h at RT in blocking solution (0.3% Triton X-100, 5% goat serum and 10% FBS in PBS). Subsequently, the cells were incubated for 2 h at RT with the primary antibodies against P2RX7 (1:100), DUSP1 (1:100), DUSP6 (1:100), or α-tubulin (1:1,000). After washing twice in PBS containing 2% BSA, the cells were incubated for 1 h at RT with appropriate Alexa Fluor™ conjugated secondary antibodies: donkey anti-rabbit IgG and goat anti-mouse IgG at a 1:500 dilution. Finally, the cells were washed in PBS, the nuclei were counterstained with DAPI, the coverslips were mounted with Aqua Poly/Mount reagent (Polysciences) and confocal images were acquired on a TCS SPE microscope with a 63× Apochromat NA = 1.3 oil objective lens (Leica Microsystems) or with an Eclipse TE2000-E microscope with a 20x Nikon Fluor NA = .45 air objective lens (Nikon). Images were quantified using the ImageJ free software.

N2a cells cultured on coverslips placed in 35 mm dishes (30,000 cels/cm2 (link)) were fixed in 4% PFA for 15 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum and 10% FBS for 1 h at room temperature. After washing with 3% BSA in PBS, cells were incubated for 1 h with primary antibodies against P2X7R (1:200), Sp1 (1:200) and/or α-tubulin (1:500). Subsequently, cells were washed with PBS and incubated for 1 h with appropriate secondary Alexa Fluor® conjugate antibodies (1:400) and nuclei were counterstained with DAPI. Coverslips were mounted using Prolong^® gold antifade reagent (Molecular Probes). Confocal images were acquired with a TCS SPE microscope from Leica Microsystems with a 63× Apochromat NA = 1.3 oil objective lens (Wetzlar, Germany) and analyzed with ImageJ software.