Alexa fluor 555 tagged donkey α goat secondary antibody

The lumbar enlargement of the spinal cord was dissected out of 6-week-old WT and Adv-KO-SCOT mice, post-fixed in 4% paraformaldehyde and soaked in 30% sucrose. 30 μm sections were blocked for two hours in Superblock, 1.5% Normal Donkey Serum, 0.5% Porcine Gelatin, and 0.5% Triton X-100 at room temperature. Sections were stained overnight with goat anti-TrkA antibody (1:250). Sections were washed with phosphate-buffered saline, stained with freshly prepared Alexa Fluor 488-tagged IB4 (10μg/mL in 1mM CaCl₂) for 10 minutes, washed again, and stained with Alexa Fluor 555-tagged donkey-α-goat secondary antibody (Molecular Probes, 1:1000) for 1 hour. Sections were coverslipped and imaged with a Nikon Eclipse Ti2 inverted microscope at 10x. The area of TrkA-positive innervation of lamina I and IB4-positive innervation of lamina IIo were quantified by ImageJ. A blinded observer manually counted the number of TrkA-positive projections extending through lamina II into lamina III or deeper. At least five images of the dorsal horn of the spinal cord were taken for each mouse, and the average TrkA⁺ and IB4⁺ areas and the number of deeper TrkA⁺-projections were used for statistical analysis.