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

Home , Phosphomolybdic acid, Silver staining, Trichrome staining

485 4.2

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 further 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 staining. 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 substances (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 dependent on environmental factors. Fixation and embedding are clearly important factors. 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 interpretation of the staining results can only be performed with consideration of the conditions. Considering that basically all morphological investigation should start with histological 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 integrity, histological stainings can also help to detect specific molecules or groups of molecules in cardiovascular tissue. For example, alteration of calcium content in infarcted 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 deposition of calcium in the extracellular space by specific staining methods (for review 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 dissolved 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 basic 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 cytoplasm 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 including the thin basement membrane of the cardiomyocytes and thin collageneous fibres (arrows). d A relatively homogeneous red staining of the sections is produced by von Kossa staining, while calcium deposits 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 preservation. 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 turn blue. 4. Immerse the sections in eosin for 30 s with agitation. 5. Wash and differentiate the sections in running tap water for about 30 s. 6. Dehydrate the sections in 70, 95% and two changes of 100% ethanol (2–3 min in each change). 7. Clear the sections in xylene and cover, using a resinous medium.

Interpretation of results

1. Nuclear chromatin should be stained blue to purple. 2. Cytoplasm, collagen, keratin and erythrocytes should be stained pink. Classical Histological Staining Procedures 489 4.2

Methylene Blue and Toluidine Blue Staining

Toluidine blue as well as methylene blue are basic dyes, which stain nucleic acids blue (the orthochromatic colour), but polyanions such as sulphated polysaccharides purple (the metachromatic colour). When dye molecules bound to sulphate groups are stacked closely together, the dye results are colour-shifted from blue to purple. Thus, a metachromatic reaction often indicates the presence of numerous closely packed sulphate groups. Therefore extracellular matrix deposits containing a large amount of proteoglycans, which contain a sulphated glycosaminoglycan side chain such as heparan, chondroitin or dermatan sulphate, can be visualized by use of metachromatic colouring (see Fig. 1b).

Both stainings are simple staining methods for resin-embedded tissue. ▬ Toluidine stock solution  1 g toluidine blue  5 g borax  100 ml distilled water  Mix and stand overnight then filter before use. ▬ Methylene blue stock solution  1 g methylene blue  1 g borax  100 ml distilled water

Method

1. Flood a section, on a hot plate, with toluidine blue or methylene blue solution for up to 30 s (do not allow the section to dry). 2. Wash in running tap water for a few seconds. 3. Remove excess water from around the section then dry on a hot plate. 4. The section can be examined uncovered or mounted if a permanent preparation is required.

Interpretation of results

1. The end result is an intense blue staining of the section. 2. Purple stained areas give evidence of metachromatic colouring.

Trichrome Staining

In the trichrome stains, which commonly employ more than one acid dye, use is made of dye competition. Consider preparations stained with a mixture of dyes of the same charge but of markedly different sizes, and so probably of different staining rates. In the early stages of staining at least, it would be expected that whilst the fast-staining tissue substrates would take up both the large and the small dyes, slow-staining substrates would be predominantly coloured by the small fast-staining dye. Taking these 4 490 Histological Techniques

Figure 2a–c Van Gieson staining is performed on normal (a) and infarcted murine myocardium (b). a Normal myocardium shows a homogeneous yellow colour in all cellular components of the myocardium, while the sparse extracellular matrix is red. b The fibrotic replacement of the myocardium can be seen as red staining of the damaged area. c At higher magnification the nearly complete loss of cardiomyocytes and the replacement by collagen fibres are recognizable

facts into account one might expect that fast-staining components such as collagen In-Vitro Techniques fibres would be coloured by the larger dye of the pair whereas slow-staining substrates such as cardiomyocytes will be coloured predominantly by the smaller dye. For example, acid fuchsin and picric acid are used in Van Gieson’s trichrome stain (Fig. 2). In the picric acid-fuchsin mixture, the small picric acid molecule reaches and stains the available sites in muscle before the larger fuchsin molecules can enter. Used by itself, acid fuchsin has no difficulty in staining muscle. The van Gieson methods give the most selective staining of collagen fibres. The 25- to 50-fold excess of picric acid relative to the collagen dye in a van Gieson solution renders the cytoplasmic background yellow. For collagen staining, different red dyes can be used such as Sirius red or acid fuchsin. If besides larger collagen fibres, reticulin fibres and basal membranes should be demonstrated, long planar molecules such as Sirius red can be used. Sirius red stains the thinnest collagen fibres, so reticulin fibres and basal membranes can also be stained (Lyon et al. 1992). Another trichrome staining method is the Goldner staining, a variant of the Masson staining. As the name implies, three dyes are employed, selectively staining muscle, collagen fibres, fibrin, and erythrocytes. The general rule in trichrome staining is that the less porous tissues are coloured by the smallest dye molecule; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule. Trichrome staining is requested if alterations in the collagen content of heart, valves and vessel wall are to be investigated (Rösen et al. 1995; Röll et al. 2002, 2002b; Ray et al. 2002; Rajamannan et al. 2003) or if structural alterations in the myocardium, as occur during early myocardial infarction, are to be investigated (Var- gas et al. 1999). Classical Histological Staining Procedures 491 4.2

Van Gieson’s Staining

Solutions

▬ Weigert’s iron-hematoxylin ▬ Stock solutions ▬ Solution A  5 g hematoxylin  500 ml 95% ethanol ▬ Solution B  5.8 g ferric chloride (FeCl3.6H2O)  495 ml water  5 ml concentrated hydrochloric acid ▬ Working s olut ion  Mix equal volumes of A and B. The mixture should be made just before using. It may be re-used many times and can be kept for 2 days at room temperature or for 10–14 days at 4 °C. Older solutions stain nuclei brown or bluish-grey rather than black (Kiernan 1999). ▬ van Gieson’s solution  0.5 g acid fuchsin  500 ml saturated aqueous picric acid  Optionally, add 2.5 ml of concentrated hydrochloric acid  This mixture can be reused many times, and it retains its staining power for more than 5 years (Kiernan 1999). ▬ Acidified water  Add 5 ml acetic acid (glacial) to 1 l of water (tap or distilled).

Method

1. De-wax and hydrate paraffin sections. 2. Stain in working solution of Weigert’s hematoxylin for 5 min (may need 10 min if the solution is more than 4–5 days old). 3. Wash in running tap water. Check the wet section with a microscope to ensure that nuclei are selectively stained. 4. Stain in van Gieson’s solution, 2–5 min. The time is not critical. 5. Wash in two changes of acidified water. 6. Dehydrate rapidly in three changes of 100% ethanol. This step also differentiates the picric acid. 7. Clear in xylene and mount in a resinous medium.

Interpretation of results

1. Nuclei should be stained black. 2. Collagen should be stained red. 3. Cytoplasm (especially smooth and striated muscle), keratin and erythrocytes should be stained yellow. 4 492 Histological Techniques

Goldner’s Trichrome Staining

Solutions

▬ Acid fuchsin, 0.5%  1.0 g acid fuchsin  200 ml water ▬ Phosophomolybdic acid-orange solution  5 g phosphomolybdic acid (molybdophosphoric acid)  5 g phosphotungstic acid  200 ml water  4 mg orange G ▬ 2.5% fast green FCF  5 g fast green FCF  195 ml water  5 ml glacial acetic acid ▬ For “Weigert’s iron-hematoxylin” see van Gieson’s Method. ▬ Acidified water with 1% acetic acid  5 ml acetic acid  1000 ml water

Method

In-Vitro Techniques 1. De-wax the sections and bring to water. 2. Stain nuclei in a working hematoxylin for 3 min. 3. Wash in running tap water for 10 min. 4. Immerse in 0.5% acid fuchsin for 5 min. 5. Wash in acidified water. 6. Immerse in the phosphomolybdic acid-orange solution for 1 min. One minute may not be enough. Therefore, check a slide under a microscope to ensure that the red dye has been removed from collagen. Return to the phosphomolybdic acid-orange solution if necessary. 7. Wash in acidified water. 8. Stain for 5 min in 2.5% fast green FCF. 9. Wash for 5 min in acidified water. 10. Dehydrate in three changes of 100% ethanol. 11. Clear in xylene. 12. Apply coverslips using a resinous mounting medium.

Interpretation of Results

1. Nuclei should be stained red. 2. Collagen should be stained blue. 3. Cytoplasm should be in various shades of red, pink and orange. 4. Cartilage matrix should be stained blue. Classical Histological Staining Procedures 493 4.2

Picro-Sirius Red Staining

Collagen forms the ground substance of connective tissue. It is composed of three amino- acids and stains strongly with acid red dyes due to the affinity of the cationic groups of the proteins for the anionic reactive groups of the acid dyes. Sirius red has the advantage of being a long, planar molecule, which becomes oriented parallel to the collagen fibre structure and increases birefringence. Red staining with picro-Sirius accompanied by a pronounced increase in birefringence is an unequivocal indication of the presence of collagen and reticulin fibres (Junqueira et al. 1979). Therefore this staining is useful if thin collagen fibres (see Fig. 1c) need to be detected in cardiovascular tissue (Van Kerckhoven et al. 2000; Ammarguellat et al. 2001) ▬ Picro-Sirius red  0.5 g Sirius red  500 ml saturated aqueous solution of picric acid  Add a little solid picric acid to ensure saturation ▬ Acidified water  Add 5 ml glacial acetic acid to 1 l of water (tap or distilled).

Method

1. De-wax and hydrate paraffin sections. 2. Stain nuclei with Weigert’s hematoxylin (see under the van Gieson method), then wash the slides for 10 min in running tap water. 3. Stain in picro-Sirius red for 1 h. 4. Wash in two changes of acidified water. 5. Dehydrate in three changes of 100% ethanol. 6. Clear in xylene and mount in a resinous medium.

Interpretation of Results

1. In ordinary bright-field microscopy collagen is red on a yellow background (see Fig. 1c). 2. Nuclei, if stained, are black.

The intensity of the red colour can be measured by microdensitometry to provide estimates of collagen content in different parts of a tissue (Kratky et al. 1996). Under examination through crossed polars the larger collagen fibres are bright yellow or orange, and the thinner ones, including reticular fibres, are green.

Silver Staining for Visualisation of Basal Membrane

Silver staining, or more precisely silver impregnation, leads to an impregnation of reticular fibres with silver salt; the fibres then appear sharply black. Collagenous fibres usually stain purple. This stain can be used with a counterstain or without, if the sil- 4 494 Histological Techniques

ver stain turns out to be very dark. Beside of the staining of reticular fibres, silver impregnation is widely used in neurohistology to stain neurons and their processes. The sections are processed in solutions containing silver, which attach to specific components in tissues. The silver is then further processed and developed into dark deposits. This staining can be used to detect alterations in the extracellular matrix, including the basal membrane, in the myocardium and vessels (Chiu et al. 1999).

Solutions

1. 0.5% periodic acid in water 2. 0.2% gold chloride in water

3. 3% sodium thiosulphate (Na2S2O3.5H2O) in water 4. Hexamethylentetramine-silversolution 100 ml 3% hexamethylentetramine 5 ml 5% silver nitrate

Dissolve 3 g hexamethylenetetramine in 100 ml water. Dissolve 250 mg silver nitrate in 5 ml of water or use 5 ml of a 5% aqueous silver nitrate stock solution. Combine the two solutions in a very clean glass bottle and mix thoroughly. A precipi- tate forms and redissolves. This solution should be stored at 4 °C as a stock solution. ▬ 50 ml hexamethylentetramine-silver stock solution In-Vitro Techniques ▬ 6 ml 5% sodium tetraborate (Na2B4O7.10H2O; borax)

This is made immediately before beginning to stain the sections.

Method

1. De-wax and hydrate paraffin sections. 2. Wash the sections in distilled water. 3. Immerse the sections in 0.5% periodic acid for 15 min. 4. Wash the sections in distilled water. 5. Place the slides in the hexamine-silver working solution for 1.5 –3 h at 50 °C in dark. 6. Wash the sections in distilled water. 7. Immerse in the gold toning solution for 2 min. 8. Wash the sections in distilled water. 9. Immerse in 3% sodium thiosulphate for 2 min. 10. Wash in running tap water for 5 min. 11. Apply hematoxylin and eosin as a counterstain if desired. 12. Dehydrate, clear and apply coverslips using a resinous mounting medium.

Interpretation of Result

Basal membrane should be stained in grey-black. Classical Histological Staining Procedures 495 4.2

Von Kossa’s Silver Staining

Tissue sections are treated with silver nitrate solution, the calcium is reduced by strong light and replaced with silver deposits, which are visualized as metallic silver. The replacement is based on inorganic precipitation, and the range of possibilities for sa- tisfactory histochemical procedures using inorganic reactions is limited. For counter- staining fast red is used. This staining leads to a black colouring of calcium-deposits (see Fig. 1d), while the cytoplasm is pink and the nuclei are red coloured. The staining can be performed on sections of formalin-fixed and paraffin-embedded tissue. The staining can be used to detect calcification in tissue, such as occurs in atherosclerosis and in cell cultures (Wada et al. 1999; Ray et al. 2002; Rajamannan et al. 2003).

Solutions

1. 1% aqueous silver nitrate (AgNO3). Solution A must be made up in the purest available water.

2. 5% aqueous sodium thiosulphate (Na2S2O3.5H2O). 3. Counterstain.

0.5% safranine or neutral red is suitable. All these solutions can be used repeatedly until precipitates form in them.

Method

1. De-wax and hydrate paraffin sections. Wash in water. 2. Immerse in silver nitrate in bright sunlight or directly underneath a 100 W elec- tric light bulb for 15 min. 3. Rinse in two changes of water. 4. Immerse in sodium thiosulphate for 2 min. 5. Wash in three changes of water. 6. Counterstain nuclei, about 1 min. 7. Rinse briefly in water. 8. Dehydrate (and differentiate the counterstain) in 95% and two changes of absolute alcohol. 9. Clear in xylene and mount in a resinous medium.

Interpretation of Results

1. Sites of insoluble phosphates and carbonates should be stained as black, brown or yellow. 2. Nuclei should be stained pink or red. 4 496 Histological Techniques

Examples Trichrome Staining for Detection of Myocardial and Perivascular Fibroses

Alterations in extracellular matrix amount and composition are found in several cardiovascular diseases such as myocardial infarction and diabetic cardiomyopathy (Rösen et al. 1995, 1998; Röll et al. 2002, 2002b). To evaluate the role of oxidative stress in the increased fibrosis in diabetic hearts, we analysed the myocardial and perivascular amounts of extracellular matrix and the composition of the extracellular matrix by Goldener trichrome staining and by immunohistochemistry against collagens I and III. Subsequent computerized morphometrical analyses of the perivascular connective tissue of coronary vessels were performed with a Leitz CBA 8000 image analysing system for detection of real colour. The area of connective tissue was given by the trichrome-stained area related to the circumference of the vessel. A good cor- relation was found between alterations in the sizes of immunohistochemically (for collagens I and III) stained areas and the changes in Goldner trichrome stained areas in the investigated groups. Considering the quality and reproducibility of trichrome staining results in cardiac tissue, the method is well suited to simple detection of connective tissue, especially if no differentiation between extracellular matrix components is requested. If a more detailed analysis of extracellular matrix composition is required, Goldner trichrome staining is an excellent control and back-up method. In the study described the trichrome result allowed us to document the beneficial role of anti-oxidants in In-Vitro Techniques diabetes. More severe alterations in the myocardium, as they occur after infarction of the heart, can be effectively shown by trichrome staining. Cellular replacement therapy represents a novel strategy for the treatment of heart failure. Before cellular replacement therapy can be used in clinical applications, a lot of questions must be answered. Therefore animal studies are required which allow further development of and prove the efficacy of this strategy. A major problem of the experimental myocardial injury models is the great varia- tion in lesion size. A relatively good reproducible lesion is induced in mouse hearts by cryoinjury. This approach instead of coronary artery ligation was chosen since the latter is known to result in large variations in infarct size in rodent hearts due to marked differences in collateralization. But variations of lesion sizes occur with the cryoinjury also. Therefore it is important to control the size of the lesion for every single experiment. Remembering that the lost myocardium will be replaced by fibrotic tissue after more than 6 days (Roell et al. 2002b), detection of fibrotic tissue by van Gieson trichrome staining is an excellent method for demonstrating the infarcted area (see Fig. 2). The subsequent use of computerized morphometric analyses allows measurement of the infarct size on cross-sections of the heart. Recently van Gieson staining was also discussed as a suitable method for detection of infarcted areas at early time points (Vargas et al. 1999). Using the van Gieson tri-chrome staining at early time points after infarction seems to be appropriate. Classical Histological Staining Procedures 497 4.2

Methylene Blue Staining of Semi-thin Sections of Araldite Embedded Tissue for Qualitative and Quantitative Morphological Analyses

Resin embedding allows the cutting of slices of less than 100 nm for electron microscopy, but also for light microscopical observation. Thin slices in combination with adequate staining procedures elicit a high structural resolution, which cannot be at- tained by other methods. Especially if microvascular vessels are to be investigated, resin-embedded and methylene blue- or toluidine blue-stained tissue has advantages as compared with paraffin-embedded material. Therefore we have chosen araldite embedding and methylene blue staining for investigation of myocardial capillaries. Semi-thin sections (0.5 mm) of perfusion-fixed myocardium allow recognition of the capillary density and the lumen width of the capillaries (Bloch et al. 1995; Tagarakis et al. 2000), if adequately stained by methylene blue. Other embedding methods do not give the good preservation of the tissue structure, which allows the high-resolution morphological investigation necessary for quantitative analysis of the capillary density and capillary lumen width. We used this method to detect maladaptation of the cardiac capillary network in a mouse exercise model evoked by anabolic steroids (Tagarakis et al. 2000). For this analysis, animals were perfusion-fixed and embedded in araldite. Methylene blue staining of 0.5 mm slices of the myocardium allowed recognition of all the myocardial capillaries, which is a prerequisite for the measurement of capillary density. Another application for araldite embedding and subsequent staining of semi-thin sections by methylene blue was the investigation of the alteration in capillary diameter in isolated perfused hearts which depends on the nitric oxide pathway. Using automated detection of capillary diameter on methylene blue stained semi-thin section of perfusion-fixed myocardium after modulation of the nitric oxide pathways in isolated perfused hearts, fast changes of capillary diameter could be investigated. It could be shown, that NO influences the dilation of the capillary microvasculature in- dependently of flow regulation (Bloch et al. 1995)

Troubleshooting Different artefacts can arise from tissue selection up to the mounting of the stained sections, which make the interpretation of the histological staining difficult. A com- mon reason for artefacts is late or poor fixation of the tissue. Late fixation allows the accumulation of acid metabolites in the tissue. Therefore the time up to the fixation of the tissue should be shortened. Another problem is that changes in volumes of cells and tissue are often observed, especially during dehydration. This may be accentuated by pathological changes. To overcome the change in volume, the osmolarity of the buffer should be optimised. Also the fixation process can lead to a shrinkage of the tissue, especially if strong cross-linking fixatives such as glutaraldehyde are used. If quantitative analyses of the amount of tissue components are required, the concentra- tion and composition of the fixative may need to be optimised. 4 498 Histological Techniques

A further problem is the loss of material from the tissues. This may be various small molecules such as peptides or ions or large molecules such as glycogen. Lipids are lost by being dissolved in the organic solvents used in processing. This can cause problems, if the content of specific molecules is being analysed. The loss of material can occur before and during fixation as well as, depending on the fixation, also at later stages of the staining procedure. False positive localization may also occur. This is found especially with soluble intercellular proteins. The use of cross-linking fixatives can reduce this problem. But problems can also be derived from the staining procedures themselves. This is mainly detected by unusual colouring of tissue structures and inhomogeneous colouring of the specimen. The protocol should then be carefully checked for the con- centrations of the dyes and the composition of the dye solution and the pH of the solution should be tested. Furthermore the differentiation times and conditions of the staining must be controlled. If all of these parameters are as described in the protocol, a control tissue, which is optimally handled, can be stained to recognize tissue- specific problems. Such tissue-specific problems can be derived from late fixation, wrong fixation conditions or inhomogeneous fixation. Detailed descriptions of troubles, which can occur in the different stainings, are found elsewhere (for example Horobin and Bancroft 1998).

References In-Vitro Techniques Ammarguellat F, Larouche I, Schiffrin EL (2001) Myocardial fibrosis in DOCA-salt hypertensive rats. Circulation 103: 319–324 Andersen AP, Prento P, Lyon H (1992) General theory for tissue staining. In: Lyon H (ed) Theory and strategy in histochemistry. Springer, Berlin, pp 283–291 Bloch W, Hoever D, Reitze D, Kopalek L, Addicks K (1995) Exogenously supplied nitric oxide influences the dilation of the capillary microvasculature in vivo. Agents and Actions [Suppl] 45: 151–156 Chatelain P, Kapanci Y (1984) Histological diagnosis of myocardial infarction: the role of calcium. Appl Pathol 2: 233–239 Chiu YT, Liu SK, Liu M, Chen SP, Lin YH, Mao SJ, Chu R (1999) Characterization and quantitation of extracellular collagen matrix in myocardium of pigs with spontaneously occurring hypertrophic cardiomyopathy. Cardiovasc Pathol 8: 169–175 Horobin RW, Bancroft JD (1998) Troubleshooting Histology Stains. Churchill Livingstone, Edinburgh Junqueira LCU, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11: 447–455 Kiernan JA (19999 Methods for connective tissue. In: Kiernan JA (ed) Histological and histochemical methods, theory and practice. Butterworth-Heinemann, Oxford, pp 144–164 Kratky RG, Ivey J and Roach MR (1996) Collagen quantitation by video-microdensitometry in rabbit atherosclerosis. Matrix Biology 15: 141–144 Lindal S, Smiseth OA, Mjos OD, Myklebust R, Jorgensen L (1986) Reversible and irreversible changes in the dog heart during acute left ventricular failure due to experimental multifocal ischaemia. Acta Pathol Microbiol Immunol Scand [A] 94: 177–186 Lyon H, Hoyer PE, Prento P (1992) Proteins. In: Lyon H (ed) Theory and strategy in histochemistry. Springer, Berlin, pp 283–291 Puchtler H, Meloan SN (1978) Demonstration of phosphates in calcium deposits: a modification of von Kossa’s reaction. Histochemistry 56: 177–185 Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T (2003) Human aortic valve calcification is associated with an osteoblast phenotype. Cir- culation 107: 2181–2184 Ray R, Choudhary SK, Kumar AS (2002) Pathological aspects of explanted homograft mitral valve. Cardiovasc Pathol 11: 177–180 Roell W, Fan Y, Stoecker E et al. (2002) Cellular cardiomyoplasty in a transgenic mouse model. Transplantion 73: 462– 465 Classical Histological Staining Procedures 499 4.2

Roell W, Lu ZJ, Bloch W et al. (2002b) Cellular cardiomyoplasty imporves survival after myocardial injury. Circulation 105: 2433–2439 Rösen P, Ballhausen T, Bloch W, Addicks K (1995) Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 38: 1157–1168

Rösen P, Hönack C, Müssig K, Bloch W, Addicks K. (1998) Influence of AT1 receptor inhibition on cardiac function and structure of diabetic rats. In: Dhalla NS, Zahradka P, Dixon I, Beamish R (eds) Angiotensin II receptor blockade: physiological and clinical implications. Kluwer Academic Publishers, Boston, pp 245–260 Tagarakis C, Bloch W, Hartmann G, Hollmann W, Addicks K (2000) Testosterone-propionate leads to maladaptation of the cardiac capillary bed to muscular exercise. Med Sci Sports Exerc 32: 946–953 Van Kerckhoven R, Kalkman EA, Saxena PR, Schoemaker RG (2000) Altered cardiac collagen and associated changes in diastolic function of infarcted rat hearts. Cardiovasc Res 46: 316–323 Vargas SO, Sampson BA, Schoen FJ (1999 Pathologic detection of early myocardial infarction: a critical review of the evaluation and usefulness of modern techniques. Mod Pathol 12: 635–645 Wada T, McKee MD, Steitz S, Giachelli CM (1999) Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circulation 84: 166–178