title: Dual use of immunohistochemistry for film densitometry and light microscopy.
abstract: In the present study, we applied the principles of immunoblotting and light microscopy immunohistochemistry to develop a combined methodology that allows obtaining optical density data in films, as well as morphological and protein distribution data on slides using the same brain tissue section, thus maximizing the data obtained from a single sample. This is especially important when experiments are performed using very valuable or unique tissue samples, which is a very common case in the study of the human brain. The ideal methodology should combine the possibility of measuring levels of expression of a marker, and the capability to map accurately the distribution of that marker in the region of interest. To achieve this, two things are required: first, the technique needs to be sensitive enough to obtain optical density or intensity measurements of the marker, and second, a good preservation of the tissue is needed for the study of distribution patterns and morphological analysis. Here we show that our combined methodology produced reliable results for different tissue preservation conditions (fresh-frozen and fixed tissue), in different species (rat and human), in different brain areas (substantia nigra and striatum), and for the detection of different markers (tyrosine hydroxylase and μ-opioid receptor). This methodology also combines the accuracy of optical density data acquisition in film with obtaining histological slides from the same sample. In summary, the methodology proposed here is very versatile and does not require the use of specialized equipment, other than the routine equipment present in an anatomy laboratory.
authors.affiliation: Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Sparks Center (SC865 C), 1720 7th Avenue South, Birmingham, AL 35294, USA. melendez@uab.edu
authors.forename: Miguel
authors.initials: M
authors.lastname: Melendez-Ferro
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Rice
authors.forename: Rosalinda C
authors.initials: RC
authors.lastname: Roberts
authors.forename: Emma
authors.initials: E
authors.lastname: Perez-Costas
mesh_terms: D004867:Equipment Design; D019544:Equipment Failure Analysis; D005373:Film Dosimetry / Q000295:instrumentation* / Q000379:methods*; D015151:Immunoblotting / Q000295:instrumentation* / Q000706:statistics & numerical data*; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D018511:Systems Integration