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Classical Histological Staining Procedures in Cardiovascular Research Wilhelm Bloch and Yüksel Korkmaz

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Classical Histological Staining Procedures in Cardiovascular Research Wilhelm Bloch and Yüksel Korkmaz 485 4.2 Classical Histological Staining Procedures in Cardiovascular Research Wilhelm Bloch and Yüksel Korkmaz Introduction The development of new methods for qualitative and quantitative morphological in- vestigations has been growing exponentially. In particular, methods allowing the detection of proteins and mRNA such as immunohistochemistry and in situ hybri- disation have been widely expanded in their use. Although these methods are more and more important in morphology, the classical staining procedures can bring fur- ther advantages compared to these methods and can be used to get basic and supple- mentary information in cardiovascular research, so it is no wonder that they remain standard methods in cardiovascular research. The classical stains cannot be replaced if various components of tissues are to be distinguished. Hematoxylin-eosin (HE) for paraffin-embedded tissue and methylene blue or toluidine blue for resin-embedded tissue are probably the most widely used. The object of histological staining is to de- monstrate tissue and cell components in their native localization by using chemically well-defined methods. The two main tissue units are the cell and the extracellular matrix, therefore the targets for histological staining are the molecular and structural components of the cells and the extracellular matrix. Histological staining often per- mits initial recognition of the molecular and structural components of the tissue. The heterogeneous chemical composition of the tissue, which leads to the molecular and subsequently to the structural composition, is a prerequisite for the histological stain- ing. The chemical components of the cell and the extracellular matrix are the direct targets for the dyes, which can be defined as chromogens of aromatic or heteroaro- matic nature, soluble in water or polar solvents and capable of binding to other sub- stances (Anderson et al. 1992). The chemical components are of inorganic (water and salts) and organic (proteins, nucleic acids, carbohydrates and lipids) nature. Proteins, nucleic acids, carbohydrates, and lipids can occur as pure substances in the tissue, but they are more frequently found as molecular complexes or mixed compounds. The different affinities of the dyes for these tissue components allow the specific labelling of cell and extracellular matrix structures and molecules. Unfortunately the staining result is not only dependent on the chemical composition of the tissue. It is also de- pendent on environmental factors. Fixation and embedding are clearly important fac- tors. Differences in the preparation of tissue will produce differences in the staining. Therefore standardization of a staining method for cytological and histological speci- 4 486 Histological Techniques mens requires consideration of all steps in the procedure; the subsequent interpre- tation of the staining results can only be performed with consideration of the condi- tions. Considering that basically all morphological investigation should start with histo- logical staining of the tissue, it seems necessary to get an overview about preservation of the tissue and the alterations that occur. It is not surprising that standard stainings such as HE and methylene blue or toluidine blue are so often used in cardiovascular research. Besides the simple detection of structural integrity or alteration of the in- tegrity, histological stainings can also help to detect specific molecules or groups of molecules in cardiovascular tissue. For example, alteration of calcium content in in- farcted myocardium can be detected by alizarin red (Chatelain and Kapanci 1984) or oil red O staining revealing deposition of fine lipid droplets in the ischemic cardiac muscle cells bordering on the necrotic areas (Lindal et al. 1986). But mostly, light microscopic histological staining is used to visualize alterations in the extracellular matrix (Rösen et al. 1995; Tagarakis et al. 2000; Röll et al. 2002) and to identify de- position of calcium in the extracellular space by specific staining methods (for re- view Puchtler and Meloan, 1978) as well as for detection of both (Rajamannan et al. 2003). This chapter can give only a small insight into classical staining procedures. Therefore the chapter is focused on the most frequently used basic staining methods and those which can be used to find alterations in the extracellular spaces of vessels and myocardium. In-Vitro Techniques Description of Methods and Practical Approach Histological staining is based on physical and chemical reactions. The following mechanisms involved in the staining process can be described. A dye, which is dis- solved in a staining solution, may be absorbed on the surface of a structure, or dyes may be precipitated within the structure, simply because environmental factors (such as pH, ionic strength, temperature) favour absorption or precipitation. Most staining reactions involve a chemical reaction between dye and stained substance through salt linkages, hydrogen bonds, van der Waals forces, coulombic attractions or covalent bonding. Staining with such dyes results in a predictable colour pattern based in part on the acid-base characteristic of the tissue. In general, the staining of tissues is also affected by the number and distribution of binding sites for dyes in the tissues. However, colour and colour distribution are not absolutely reliable for discrimination between tissue components. Colour will vary with the specific stain used and also with the conditions that exist during preparation of the slide. These include everything from the initial fixing solution to the ionic strength of the staining solution and the differ- entiating solvents utilized after staining. The dyes most used for histological staining can be subdivided into acidic and ba- sic dyes. An acid dye exists as an anion in solution, while a basic dye exists as a cation. Often the staining solutions are composed of basic dyes and acidic dyes, as for ex- Classical Histological Staining Procedures 487 4.2 ample the widely used hematoxylin-eosin staining, where the hematoxylin-metal complex acts as a basic dye and the eosin as an acidic dye. This allows detection of different structures in a specimen dependent on their charge. A further commonly used staining mechanism, called trichrome staining, is a dye competition technique, which allows tissue component-specific staining with dyes of different molecular weight. In the following, staining methods, such as HE, methylene blue, trichrome stainings, Sirius red, silver impregnation and von Kossa, often used in cardiovascular research are described in more detail. Hematoxylin and Eosin Staining (HE) H.E. is a good general stain and is therefore the most used. It is a staining method which uses a basic and an acidic dye. A hematoxylin-metal complex acts as a basic dye, staining nucleic acids in the nucleus and the cytoplasm blue, brown, or black. Eosin, an acid aniline, stains the more basic proteins within cells (cytoplasm) and the extra- Fig. 1a–d Histological staining of paraffin (a, c, d) and araldite-embedded murine heart tissue (b). a Micrograph section stained with hematoxylin-eosin. Note the dark blue to purple staining of nuclei and the pink staining of the cytoplasm. b Methylene blue stained semi-thin sections reveal a blue staining of cyto- plasm and nuclei, which are only distinguished by colour intensity. Metachromatic colouring of the extracellular matrix leads to a blue to lilac staining of the extracellular matrix (asterisk). c Sirius red staining produces a yellow colouring of the cells and a red staining of the extracellular matrix includ- ing the thin basement membrane of the cardiomyocytes and thin collageneous fibres (arrows). d A rela- tively homogeneous red staining of the sections is produced by von Kossa staining, while calcium de- posits in a heart valve are black (arrows) 4 488 Histological Techniques cellular matrix pink to red (Fig. 1a). In myocardium, the nuclei are blue-black, while the muscle fibres are pink to red. The staining is strongly dependent on the acidic or alkaline conditions. Under acidic conditions, proteins have a net cationic charge and have an affinity for the basic dyes. Under alkaline conditions, basic dyes will stain all the tissue and selectivity will be lost. Rates of dye binding and loss may vary between different structures in a tissue, thus aiding differentiation. HE staining can be used for frozen and chemically fixed tissues by appropriate changes in the staining procedure. A detailed staining procedure is only given for formaldehyde-fixed and paraffin-embedded tissue, which show good structural pres- ervation. HE staining is a basic method usable for nearly all morphological investiga- tions in cardiovascular research to get information about the structure of the tissue. Solutions ▬ Mayer’s haemalum Dissolve the following, in the order given, in 750 ml of water 50 g Alumminium potassium sulphate [KA(SO4)2 12H2O] 11.0 g Haematoxylin 0.1 g Sodium iodate (NaIO3) 1.0 g Citric acid (monohydrate) 50 g Chloral hydrate ▬ Eosin 2.5 g eosin In-Vitro Techniques 0.5 ml glacial acetic acid 495 ml water ▬ Acid-alcohol 500 ml 95% alcohol 5 ml concentrated hydrochloric acid Method 1. De-wax and hydrate paraffin sections. Frozen sections should be dried on to slides. 2. Immerse the sections in Mayer’s haemalum for 1–15 min (usually 2–5 min, but this should be tested before staining a large batch of slides). 3. Wash in running tap water for 2–3 min or until the sections
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