Fitc conjugated anti mouse secondary antibody

Frozen DRG sections (8 µm) were fixed with 4% paraformaldehyde for 30 min. Following blocking with 5% bovine serum albumin, the DRG sections were incubated with anti-TNF-α (Millipore; 1∶100) and anti-GAP43 (Millipore; 1∶100) antibodies overnight at 4°C. Following 3 washes in 1× PBS, the sections were incubated with an anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Molecular Probe; for TNF-α, 1∶200) or an anti-mouse FITC-conjugated secondary antibody (Dako, for GAP-43, 1∶200) for 1 hour at room temperature. The sections were then washed twice in 1× PBS and incubated for 5 min with 4′,6-diamidino-2-phenylindole (DAPI; 10 µg/ml; Sigma) for nuclear counterstaining, followed by 3 additional washes with 1× PBS. Finally, the sections were dried, mounted and observed through fluorescence microscopy (Nikon, Eclipse, TE300). After staining, images of samples from three independent experiments were captured on a fluorescence microscope. Ratios of fluorescent signal were measured from three random of microscopic areas (18548.61 µm² per field) [23] (link). Images were captured using a digital camera (EvolutionTM VF) and analyzed with Image-Pro Plus® software (Media Cybernetics, Silver Spring, MD).

After fixation in 4% paraformaldehyde, slides were washed twice in PBS, and further incubated with 0.2% Triton X-100, 3% BSA in PBS for 30 minutes at room temperature for blocking and permeabilization. Paxillin was visualized using the mouse monoclonal antibody clone 5H11 purchased from Millipore (dilution 1:100 in PBS) for 1h at room temperature, followed by the anti-mouse FITC-conjugated secondary antibody (DAKO, Glostrup-Denmark, dilution 1:100 in PBS) for 1h at room temperature. The actin cytoskeleton was visualized by staining with TRITC-phalloidin, as previously described [6 (link)]. Nuclei were counterstained with DAPI (Vector Laboratories Inc, Burlingame, CA, USA). Slides were mounted and viewed using a Leica DMI 6000 B, equipped with Hamamatsu ORCA-R2 digital camera. Cell area was measured using ImageJ software (http://rsbweb.nih.gov/ij/).