Notre Dame Integrated Imaging Facility

Protocols

 

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.

Wright and Giemsa stains are Romanowsky stains used to stain peripheral blood and bone marrow smears. The most important components of these stains are oxidized methylene blue, azure B and eosin Y dyes. The eosin Y dye stains the cytoplasm of cells an orange to pink color. The methylene blue and azure B dyes stain the nucleus varying shades of blue to purple. Romanowsky stains are used to perform differential white blood cell counts and to study red blood cell morphology. Abnormal granulocyte, lymphocyte or monocyte cell counts may be used to facilitate the diagnosis of diseases such as leukemia or bacterial infections.

This method is used for detection of glycogen in tissues such as liver, cardiac and skeletal muscle on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen sections as well. The glycogen, mucin, and fungi will be stained purple and the nuclei will be stained blue.

This method is used for the detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red.

Mast cells are found in the connective tissue and their cytoplasm contains granules (metachromatic) composed of heparin and histamine. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining). Metachromasia, tissue elements staining a different color from the dye solution, is due to the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements.

Cryofixation, cryosubstitution and cryoembedding are a set of low-temperature methods for immunocytochemical and microanalytical ultrastructural studies.  Sections prepared by these three cryomethods can be used to determine elemental composition, molecular compositions, functions and 3-D ultrastructure. The information obtained can be treated by multivariate statistical methods. Thus, each cellular compartment can be identified by its morphology, molecular and elemental composition and function and changes in these data during physiological and pathological processes can be monitored.

Most of the newly developed microscopic techniques make use of fluorescence. Microscope and accessories performance is also increasing in accordance with the requirements of these applications and the fast growing number of fluorochromes available.

DAPI is a popular nuclear counterstain for use in multicolor fluorescent techniques. Its blue fluorescence stands out in vivid contrast to green, yellow, or red fluorescent probes of other structures. When used according to our protocols, DAPI stains nuclei specifically, with little or no cytoplasmic labeling. The counterstaining protocols are compatible with a wide range of cytological labeling techniques—direct or indirect antibody-based detection methods, mRNA in situ hybridization, or staining with fluorescent reagents specific for cellular structures. DAPI can also serve to fluorescently label cells for analysis in multicolor flow cytometry experiments.