Goat anti rabbit rhodamine conjugated secondary antibody

Living mixed cortical cells were stained for 1 h at 4 °C using rabbit polyclonal anti-PKR₁ or anti-PKR₂ antibodies 1:400 in PBS (Alomone Labs, Jerusalem, Israel), washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (Sigma) for 30 min at room temperature. This procedure allowed us to specifically target surface protein expression. Cells were then fixed in 4% (w/v in PBS) paraformaldehyde for 10 min at room temperature on immunofluorescence-labeled coverslips. For PROK2 immunofluorescence, CNs were first fixed in 4% (w/v in PBS) paraformaldehyde following the above procedure, incubated with rabbit polyclonal anti-PROK2 antibody (Abcam, Cambridge, UK 1:400), washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (Sigma) for 30 min at room temperature.
CNs cells were then incubated with mouse anti-NeuN (Sigma, 1:200 dilution) or mouse anti-GFAP (Sigma, 1:400 dilution) overnight at 4 °C and with a goat anti-mouse alexa-488 conjugated secondary antibody (Sigma, 1:400) for 30 min at room temperature. For nuclei visualization, coverslips were incubated with Hoecsht 33258 (0,25 μg/ml) for 5 min at room temperature. Cells were visualized by a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Final figures were assembled by using Adobe Photoshop 7 and Adobe Illustrator 10.

Experiments were carried out as specified previously.^{52 (link)} Briefly, CGNs were fixed with 4% paraformaldehyde for 15 min and then incubated in 1% bovine serum albumin, 10% normal goat serum, 0.3 M glycine in 0.1% phosphate-buffered saline (PBS)-Tween for 1 h to permeabilize them and block nonspecific protein–protein interactions. Cells were then incubated with the primary polyclonal antibody raised against NK1 receptor (1 : 400 in PBS; Abcam, Cambridge, UK) or NeuN (1 : 200; Sigma-Aldrich) overnight at +4 °C, and then washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (1 : 1000; Sigma-Aldrich) for 30 min at room temperature. Cells were also stained with Hoechst 33258 (250 ng/ml) for 5 min at room temperature to label neuronal nuclei. Neurons were then visualized by fluorescent microscopy.^{52 (link)}