In situ localization of plethodontid courtship pheromone mRNA in formalin-fixed tissue

Male plethodontid salamanders produce courtship pheromones that increase female receptivity. Three protein components of the courtship pheromone cocktail have been characterized in the mental gland of Plethodon shermani, the red-legged salamander: plethodontid receptivity factor (PRF), plethodontid modulating factor (PMF), and sodefrin precursor-like factor (SPF). In this study, a streamlined in situ hybridization (ISH) protocol, employing a biotinylated oligonucleotide probe, is used to visualize the sites of pheromone expression in formalin-fixed paraffin-embedded P. shermani mental gland and post-cloacal tail tissue. Results corroborate previous RT-PCR studies on pheromone expression. PRF and PMF are highly expressed in P. shermani mental gland, while SPF expression is more variable. None of the tested pheromones is expressed in dorsal or ventral tail glands. The reported protocol is simple, rapid, and effective, allowing visualization of high-copy mRNA transcript in formalin-fixed tissue.