title: Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.
abstract: The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.
authors.affiliation: Ponce Health Sciences University-Medical School and Ponce Research Institute, PO Box 7004, Ponce, PR, 00732-7004, USA. tonyisidro@me.com.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Isidro
authors.affiliation: Ponce Health Sciences University-Medical School and Ponce Research Institute, PO Box 7004, Ponce, PR, 00732-7004, USA.
authors.forename: Angel A
authors.initials: AA
authors.lastname: Isidro
authors.affiliation: Ponce Health Sciences University-Medical School and Ponce Research Institute, PO Box 7004, Ponce, PR, 00732-7004, USA.
authors.forename: Myrella L
authors.initials: ML
authors.lastname: Cruz
authors.affiliation: Ponce Health Sciences University-Medical School and Ponce Research Institute, PO Box 7004, Ponce, PR, 00732-7004, USA.
authors.forename: Siomara
authors.initials: S
authors.lastname: Hernandez
authors.affiliation: Ponce Health Sciences University-Medical School and Ponce Research Institute, PO Box 7004, Ponce, PR, 00732-7004, USA.
authors.forename: Caroline B
authors.initials: CB
authors.lastname: Appleyard
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000276:immunology*; D008297:Male; D051379:Mice; D016612:Paraffin Embedding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D016707:Tissue Fixation
chemical_list: D000911:Antibodies, Monoclonal; D005557:Formaldehyde
keywords: Biotinylated; Double stain; IHC; Ki-67; Macrophage; Rat