Alexa fluor 488 conjugated neuronal class 3 β tubulin mouse monoclonal antibody

Frozen sections (7 µm) were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin at room temperature. The samples were incubated with primary antibodies overnight at 4°C and subsequently with corresponding secondary antibodies incubate for 1 hour at room temperature. All samples were counterstained with 4′,6-diamidino-2-Phenylindole (Sigma-Aldrich), and images were captured with an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan). Corneal whole-mount immunofluorescence staining was performed as previously described.²⁴ (link) Mouse eyeballs were collected and fixed in Zamboni's fixative for 1 hour, then the cornea was dissected around the scleral–limbal region and blocked by phosphate-buffered saline with 0.1% Triton X-100, 2% goat serum, and 2% bovine serum albumin for 2 hours, and subsequently incubated in the same incubation buffer with Alexa Fluor 488 conjugated neuronal class III β-tubulin mouse monoclonal antibody (Merck-Millipore, Darmstadt, Germany) overnight at 4°C. After washing for five times, the flat mounts were imaged on an LSM880 Zeiss inverted microscope (Carl Zeiss Meditec, Jena, Germany). The quantification of corneal innervation was calculated as the percentage of area positive for β-tubulin staining as previously described.²⁵ (link)^,26 (link)