Rabbit anti human phospho erk1 2 antibody

Mice were deeply anesthetized with isoflurane and perfused with 4% paraformaldehyde. Lumbosacral spinal cord segments were dissected, embedded in gelatin (10% in 0.1M PB), post-fixed for 4 hrs in 4% paraformaldehyde, and cryoprotected overnight (30% sucrose) at 4°C. Transverse spinal cord sections (50 μm) were cut on a microtome. Following 3×5 min washes in 0.1M PB, floating spinal cord sections were incubated for 2 hrs at room temperature in a blocking reagent (5% normal horse serum and 0.1% Triton X-100 in 0.1M PB), then overnight at room temperature in rabbit anti-human phospho-ERK1/2 antibody (1:4000 in blocking reagent; Cell Signaling Technology, Inc). Sections were washed, incubated for 2 hrs at room temperature in Cy3-conjugated anti-rabbit IgG secondary antibody (1:500), washed, mounted and coverslipped. Photographs were taken at 20× using Leica Application Suite (Leica Microsystems Inc.) software. The total number of pERK-positive cells in regions receiving input from bladder afferent neurons (i.e., superficial dorsal horn, sacral parasympathetic nucleus, and dorsal commissure ^{5 (link), 6 (link), 36 (link)}) was counted in 9–13 sections per mouse by an experimenter blinded to treatment. The average for each mouse was calculated and used for statistical analysis.