Cy2 conjugated affinipure donkey anti rabbit secondary antibody

The medium was removed from the in-vitro-cultured collagen gels and each gel culture was fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Next, the gel cultures were washed with phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 in PBS (PBT) for 10 min. Then, cells were incubated in a blocking solution (containing 1% BSA and 1% NGS in 0.2% PBT) for 45 min at RT. Next, DRG neurons were incubated with mouse anti-neurofilament H (NF-H) for 1.5 hr at RT, and PC12 cells were incubated with a rabbit antibody to α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1.5 hr at RT. The cultures were rinsed 3 times with PBS and incubated for 45 min at RT with Alexa Fluor 488 conjugated Donkey anti-mouse IgG H&L for DRG neurons, or with Cy2-conjugated AffiniPure Donkey Anti-Rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for PC12 cells. Following incubation, the cultures were rinsed with PBS. Fluorescence images were acquired by using either a Leica SP8 scanning confocal microscope, driven by LasX software, or Leica DMI8 wide-field microscope. Images from the wide-field microscope were tiled and stitched using the LasX acquisition software.

PC12 cells were fixed with 4 % paraformaldehyde for 15 min at room temperature, washed with PBS and permeabilized with 0.5 % Triton X-100 in PBS (PBT) for 10 min. Cells were then incubated in a blocking solution (containing 1 % bovine serum albumin (BSA) and 1 % normal goat serum (NGS) in 0.25 % PBT for 45 min. Next, cells were incubated with a rabbit antibody to α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C. The cells were rinsed with PBS and incubated for 45 min at room temperature with Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After incubation, cells were rinsed with PBS and mounted in an aqueous mounting medium.