A combined histochemical and double immunohistochemical labeling protocol for simultaneous evaluation of four cellular markers in restenotic arteries

We designed an effective quadruple staining protocol that combines histoche mistry (HC) and double-labeling immunohistochemistry (IHC:IHC) to stain sim ultaneously several different morphological features and cell types in vasc ular lesions. Morphometric image analysis to quantitate vascular wall thick ening, lumen area, and proliferating smooth muscle cells on consecutive ser ial sections in adequate, but morphometric precision and dependable cellula r characterizations and co-localization could be obtained if analyses are p erformed on one tissue section. The development of a neointima in the rat c arotid artery was induced by angioplasty with a balloon catheter. Tissues w ere stained for elastin by a modified van Gieson method, then processed for double-labeling IHC:IHC for proliferating cell nuclear antigen and smooth muscle actin staining followed by hematoxylin staining. The four resulting tissue stains labeled elastin filaments black, proliferating nuclei brown, smooth muscle actin red and nonproliferating nuclei blue. Our staining prot ocol improved the descriptive and quantitative analysis of relation between smooth muscle cell proliferation and protein expression. Also, neointimal thickening could be measured to analyze its relation to cellular proliferat ion. Providing one slide with four stains maximizes the information from a single slice of tissue, reduces slide preparation and analysis time, and ov ercomes the restriction of tissue sample availability. This technique can b e applies to a wide spectrum of morphologic and morphometric studies.