Rabbit anti pcna antibody

Paraffin-embedded femur and tibia samples were sectioned at 10 μm and subjected to immunostaining for PCNA. Antigen retrieval was performed on the sections with IHC-Tek Epitope Retrieval solution (IHC World, Woodstock, MD, USA) at 70°C for 20 minutes. Sections were blocked in 1% Donkey IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) with 0.2% ovalbumin (Sigma-Aldrich). Rabbit anti-PCNA antibody (1:400; Cell Signaling Technology, Danvers, MA, USA) was applied over-night at 4°C, followed by biotinylated anti-Rabbit IgG (Abcam, Cambridge, UK) and peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) incubation, and visualized by diaminobenzidine substrate (DAB; Life Technologies, Carlsbad, CA, USA). PCNA-positive cells were counted using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

For immunostaining, the surface cortical region of the brain was dissected and placed in 1X PBS overnight in preparation for whole mount staining. Whole mounts were subsequently blocked in 2% Fish gelatin (Sigma Aldrich, St. Louis, MO) with 0.1% Triton X-100 for 3 h then incubated overnight in antibodies against smooth muscle actin (SMA) (1:1000; Abcam, Cambridge, UK; ab7817) in blocking buffer at 4 °C. Whole mounts were washed 5 times with 1X PBS then incubated with anti-mouse Alexa Fluor 488 conjugated secondary antibody (Molecular Probes, Carlsbad, CA) overnight at 4 °C. After washing 5× with 1X PBS, whole mounts were counterstained with DAPI (ThermoFisher Scientific, Waltham, MA) for 5 min, washed 3× with 1X PBS and embedded in mounting media (SouthernBiotech, Birmingham, AL) in a 35 mm glass dish, cover slipped, then imaged on an inverted Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany). For PCNA staining, whole mounts were incubated in HCL for 45 min at 37 °C and then neutralized with sodium borate as previously described (Kennedy et al., 2000 (link)). This method is used to unmask the PCNA antigen. Tissue was blocked then incubated in rabbit anti-PCNA antibody (1:1000; Cell signaling, Danvers, MA) overnight, and then incubated with anti-rabbit 488 (Invitrogen, Carlsbad, CA) secondary antibody. The contralateral hemisphere was used as a negative control.