Protocol

Hematoxylin and Eosin Staining of Tissue and Cell Sections

This protocol was adapted from "Preparation of Cells and Tissues for Fluorescence Microscopy," Chapter 4, in Basic Methods in Microscopy (eds. Spector and Goldman). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

Hematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.