Anti rabbit cy2 conjugated antibody

Following incubation for the designated number of days, coverslips with adherent BMM were harvested and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature and washed three times with PBS. BMM were permeabilized with 0.01% Triton X in PBS for 10 minutes at room temperature. Cells were incubated overnight at 4°C with goat anti-mouse gp91-phox and rabbit anti-mouse p67-phox (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1∶50 and 1∶100 dilution in PBS, respectively. After incubation, coverslips were washed three times with PBS and incubated for 1 hour at room temperature with anti-goat Cy3-conjugated antibody or anti-rabbit Cy2-conjugated antibody, respectively (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1∶200 dilution in PBS. BMM were counterstained with 4′6-Diamidino-2-phenindole (DAPI) according to manufacturer's instructions (Sigma, St. Louis, MO). Coverslips were mounted onto slides using MOWIOL (Calbiochemical, La Jolla, CA) and viewed by sequential scanning confocal microscopy using an Olympus IX81 inverted scope (Olympus America Inc., Center Valley, PA). Quantitative co-localization analysis was performed with Olympus Fluoview version 2.1b software. A Pearson's correlation coefficient was calculated for channel 1 and channel 2 pixel intensity for each of >100 cells per treatment group and then averaged.

Hippocampal neurons were washed twice in cold 0.01 M of PBS (pH 7.4) and fixed at room temperature in 4% of paraformaldehyde for 20 min. Following three washes in PBS, cells were blocked in PBS containing 3% of BSA for 30 min, and then incubated overnight at 4 °C with the rabbit anti-K_V2.1 antibody (1:1000, Alomone Labs, Jerusalem, Israel). The control for the specificity of the antibody against K_V2.1 was performed with its replacement with normal serum as previously described [50 (link)]. Then, cells were washed with PBS and incubated with anti-rabbit Cy2-conjugated antibody (1:200; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature under dark conditions. Cells were finally incubated for 5 min with Hoechst 33342. Cover glasses were mounted with a SlowFade Antifade Kit (Molecular Probes, Life Technologies, Milan, Italy) and acquired by a 63× oil immersion objective using a Zeiss inverted 700 confocal microscope.

Cells were cultured on glass coverslips until of a suitable density and were fixed in 4% paraformaldehyde. The cells were permeabilised in 0.3% Triton x-100 and non-specific staining was prevented by incubation in 10% goat serum in PBS. The primary antibody, p27^Kip1 (C-19, Santa Cruz) was applied for 1 hour at room temperature, at a concentration of 1:100. The cells were washed and Cy2-conjugated anti-rabbit antibody (Jackson) was applied for 1 hour at room temperature, at a concentration of 1:200. The cells were washed and the coverslips were mounted using ProLong Gold Antifade reagent (Life Technologies) with DAPI. Slides were viewed using an Olympus BX51 upright microscope using a 40x 0.75 UPlanFLN objective and captured using a Coolsnap EZ camera (Photometrics) through MetaVue Software (Molecular Devices). Filter sets for DAPI (31000v2) and FITC (41001) were used. Images were processed and analysed using ImageJ (http://rsb.info.nih.gov/ij). To quantify nuclear stains of p27^Kip1, 5 random fields were imaged and the colour channels were separated. A minimum total of 1000 nuclei were counted per cell line (based on DAPI staining).