Histochemistry

Jinsong Zhou, Xi'an Jiaotong University Press Co.

• Publisher: De Gruyter
• Year: 2017
• ISBN:
  9783110531817
• Edition: 1
• Topic: Sciences biologiques
• Subtopic: Biotechnologie

This book systematically illustrates theories and technologies in Histochemistry, including different kinds of enzymes, immunohistochemistry, polymerase chain reaction, related electron microscopic cytochemical techniques as well as the quantitative assay metrology. Abundant experiments as well as vivid images are demonstrated, making the book an essential reference for both graduate students and researchers in biochemistry.

1Introduction

The development of life sciences requires deep understanding about the chemical compositions and their structural and functional relationships. During the past hundreds of years, more and more theories, technologies and methods were found and created to detect the chemical compositions in tissues and cells, and a borderline discipline, histochemistry, was established. As a methodology, nowadays histochemistry is widely used in basic life science studies and clinical application.

1.1The History, contents and theories in histochemistry

1.1.1Origin and development of histochemistry

Histochemistry is also known as microchemistry, which means the reaction processes and results will be observed not with naked eyes or in the test tube but under a microscope. At the beginning, the chemistry methods just occupied a little in histochemistry contents because of very few chemistry knowledge. In the 20th century, those eager to know the nucleic acids, the proteins and the enzymes require the theories and methods of histochemistry to develop very fast; thus, more and more new methods were created and verified. For example, the Feulgen reaction and the methylene green-pyronine were used to display the nucleic acid, PAS reaction was introduced to show glycogens and the calcium-cobalt and lead nitrate methods were applied to demonstrate the alkaline phosphatase and acid phosphatase. The immunohistochemistry method was created when specific antigen-antibody reaction was used to detect the antigen compositions in tissues and cells, and the molecular biology technologies, such as in situ hybridization and polymerase chain reaction (PCR), were introduced and combine with histochemistry. In the meantime, the quantitation assay technologies, such as photomicrography, image analysis, flow cytometry and laser scanning confocal microscopy, were established and taken in application.

Thus, as a newly established borderline discipline, histochemistry knowledge contains the information in histology and its related lasted methods, advanced technologies and discoveries. Thus, the concept of histochemistry should be like this: Based on histology, the modern methods and technologies in physics, chemistry, biochemistry, immunology and molecular biology are introduced to detect the chemical compositions in situ, and the qualitative and quantitative analysis are conducted to understand the normal and abnormal rules about the metabolisms, functions and morphology changes in cells and tissues.

1.1.2Contents of histochemistry studies

1.1.2.1Inorganic materials in cells

These inorganic materials in cells and tissues mainly include the different kinds of metal ions, such as Ca2+, Mg2+, K+, Zn2+, Fe2+, Fe3+, Cu2+, Pb2+, Ag+, Au3+, As3+, U6+ and I–, and their salts, such as chloridate, phosphate, carbonate and nitrate.

1.1.2.2Organic materials in cells

These organic materials in cells and tissues mainly include sugar (such as glycogen, starch, glycoprotein and proteoglycan), lipid (such as phospholipid, glycolipid, lipoprotein, lipoid, cholesterol and sterol ester), nucleic acid (such as DNA and RNA), peptide, protein, pigments and different kinds of vitamins.

1.1.2.3Different kinds of enzymes

As special proteins, the enzymes play important roles in cell metabolism. Today, more than 200 kinds of enzymes can be displayed by histochemistry methods, such as acid phosphatase, alkaline phosphatase, 5-nucleotidase, glucose-6-phosphatase, adenosine triphosphatase, adenosine triphosphatase, carbonic anhydrase, nonspecific esterase, cholinesterase, cytochrome oxidase, peroxidase, monoamine oxidase, succinate dehydrogenase, lactate dehydrogenase, 3b-hydroxy steroid dehydrogenase, acylase and phosphorylase.

1.1.2.4Antigen and antibody in cells and tissues

The specific antigen-antibody reaction can be used to detect not only the antigens in tissues and the antigen from pathogenic microorganism but also the autoantibody and antibody-antigen complex to provide reliable results for basic research and clinic diagnosis.

1.1.2.5Endogenous and exogenous gene segments

With or without PCR technology, the in situ hybridization technology can be used to detect endogenous gene segments and their normal and abnormal expression, such as DNA, mRNA and the gene segments of virus, which can be used as references for clinical diagnosis.

1.1.3Theories of histochemistry

1.1.3.1Chemistry reaction

The already-known chemical reactions are used to demonstrate the materials in tissues and cells by forming colorful deposits or high electron density structures in one or several steps, such as the enzyme histochemistry. Alternatively, first, the chemicals used for detection will be structurally changed in situ by the need-to-know materials to be suitable for subsequent reactions, and second, the additional chemical regents are applied to indirectly demonstrate the need-to-know materials in cells and tissues, such as PAS and Feulgen reaction.

1.1.3.2Physics theory

The materials can be visualized by their physics characteristics, for example, the fat can be shown by colorful Sudan series dyes because the dyes are lipid-soluble, and the florescence can be observed when monoamine (such as noradrenaline, dopamine and 5-hydroxytryptamine) is induced by formaldehyde under the fluorescence microscope.

1.1.3.3Biological characteristic

Most biomacromolecules can be regarded as antigens, which will be combined and visualized by the specific antibodies that are marked by fluorescence, enzymes or colloid gold, and this is the basic theory of the widely used immunohistochemistry. Some affinity chemical reactions, for example, the reaction between avidin and biotin, forms a branch of histochemistry, the affinity histochemistry.

1.1.3.4Nucleotide chain complementary theory

The two complementary nucleotide chains will combine with each other to form stable hybrid. When one of them is labeled by a marker, it can be used in the in situ hybridization to show another (target) chain.

1.2Basic requirements of histochemistry methods

The different theories, procedures and results match different histochemistry methods, but they all have the same purpose: to display the tissue and cell chemicals in situ. Thus, they must follow the basic common requirements.

1.2.1Basic requirements

1.2.1.1Specificity

The high specificity of reactions against the need-to-know materials guarantees the right experiment results.

1.2.1.2Sensitivity

The high sensitivity of reactions makes sure that the method can be used to detect trace need-to-know materials in cell and tissue.

1.2.1.3Fixation

The perfect fixation will provide with almost an authentic cell, tissue and material structures before histochemistry staining, which is necessary for in situ observation and records.

1.2.1.4Reaction deposits

The reaction deposits must be formed in situ, insoluble, stable and colorful for light microscopy or with high electron density for electronic microscopy.

1.2.1.5Repeatability

The repeatability is a basic rule for all kinds of scientific studies.

1.2.2Things you need to know

a.The characteristics of need-to-know materials, such as water solubility, lipid solubility and the possible location in cell and tissue, must be clear before experiment. This means many references must be thoroughly read and understood.

b.The conditions of each procedure in experiments must be strictly controlled, such as the concentration of each kind of chemical reagent, the temperature and the pH value of reaction liquor, especially for the enzymes. In the control test and following repeated experiments, the conditions should remain the same.

c.The control t...