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 histochemistry (HC) and double-labeling immunohistochemistry (IHC:IHC) to stain simultaneously several different morphological features and cell types in vascular lesions. Morphometric image analysis to quantitate vascular wall thickening, lumen area, and proliferating smooth muscle cells on consecutive serial sections in adequate, but morphometric precision and dependable cellular characterizations and co-localization could be obtained if analyses are performed on one tissue section. The development of a neointima in the rat carotid artery was induced by angioplasty with a balloon catheter. Tissues were 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 protocol 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 proliferation. Providing one slide with four stains maximizes the information from a single slice of tissue, reduces slide preparation and analysis time, and overcomes the restriction of tissue sample availability. This technique can be applies to a wide spectrum of morphologic and morphometric studies.