Cell science
Notes on the development of microscopy, techniques in histology and methods for the preparation of classical staining solutions and reagents. Our focus is on immunohistological and molecular-specific principles for the visualization of biomarkers. Application examples emphasize their usefulness for the study of cellular differentiation in ontogenesis, cancer research and histopathology.
Microscopy, histology
Innovative methods provide insights into structural details. Complex methods enable high specificity.
- Immunohistology: an introduction to selective cell labeling as an essential element in the study of structure-function relationship
- The microscope as a scientific tool
- Fluorescence and fluorescence microscopy
- Histological staining techniques
- Dyes, stains, and special probes in histology
- Immuno-enzyme techniques in cytochemistry
- General aspects in tissue preparation
- Fixation of biological specimens
- Tissue dehydration, embedment
- Cryostat, cryotechniques
- Microtomy of tissue specimens, collection of sections
Antibodies, marker molecules, conjugation
Immunohistological cell labeling requires specific antibodies. Moreover, the detection of antigen-antibody reactions in tissue is based on the use of marker molecules with defined physico-chemical properties. Suitable marker molecules are, for example, fluorochromes and enzymes as reporters of selective binding reactions.
- Antibody molecules and the antigen-antibody interaction
- Immunization, immune sera and the production of antibodies
- Specificity of antibodies
- Purification of antibodies by use of biochemical and immunochemical techniques
- Immunohistological marker molecules
- Fluorochromes in selective cell analysis
- Enzymes as marker molecules
- Markers for immuno-electron microscopy
- Ferritin as electron dense label
- Gold labelled reagents as marker system
- Preparation of conjugates
Selective cell staining
Biomarkers can be visualized in histological preparations using direct or indirect labeling principles. The appropriate format is selected from testing a variety of options. Generally, the best format must be adapted to the specific requirements. This concerns both the properties of the organ tissue and the molecules to be detected.
- Cell staining with direct and indirect assay formats
- Considerations prior to immuno-staining
- Retrieval of antigenic determinants
- Inhibition of endogenous enzyme activity
- Staining enhancement, signal amplification
- Lectins and nucleic acids as molecular probes
- Immuno-staining of paraffin embedded tissue sections
- Immuno-staining of resin embedded tissue sections
- Preembedding immuno-staining of specimens for electron microscopy
- Immunofluorescence staining of cryostat sections
- Immunofluorescence assay for autoantibodies to nuclear antigens (ANA) on HEp-2-cells
- Specificity and standardization of immunohistology
- Practical aspects in quality control of immunohistology
Reagents, methods
Selection of frequently used reagents and solutions for immunohistology, cytochemistry and biochemistry. The purity of the reagents is the key for the quality of results.
- Fixatives
- Buffer solutions
- Washing solutions
- Blocking solutions
- Incubation and antibody dilution buffers
- Enzyme cytochemical substrate solutions
- Natural and synthetic dyes in histology
- Conjugation of antibodies by use of glutaraldehyde
- Protein labeling with biotin derivatives
- Preparation of soluble PAP complexes
- Measurement of peroxidase (HRP) enzyme activity in conjugates
- Measurement of glucose oxidase (GOD) enzyme activity in conjugates
- Dehydration and paraffin embedding of tissues
- Dehydration an resin embedment of tissues
- Silane conditioning of glass slides
- Elimination of mercury salts from tissue sections
- Peroxidase (HRP) cytochemistry
- Glucose oxidase (GOD) cytochemistry
- Haematoxylin staining methods
- Carmine staining methods
- Gallocyanin-chrome alum method
- Methyl green counterstaining
- Toluidine blue staining
- Romanowsky_Giemsa staining
- Periodic acid-Schiff (PAS) reaction
- Staining with Alcian blue at pH 2.5
- Azure-methylene blue staining of sections from resin embedded tissues
- Acronyms and terms in cell research and microscopy
Applications in cell science
Examples of investigations of biomarkers in tissue sections for the localization of oncofetal proteins (alpha-fetoprotein), enzymes and glycoconjugates in experimental models and in histopathological preparations.
- Cellular fluctuations, alpha-fetoprotein expression, and the question of stem cells in experimental liver regeneration and hepatocarcinogenesis
- Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury
- Immuno-electron microscopy of alpha-1-fetoprotein during normal development of rat hepatocytes
- Ultrastructural detection of alpha1-fetoprotein in hepatomas by use of peroxidase-labelled antibodies
- Focal elevation of liver microsomal epoxide hydrolase in early preneoplastic stages and its behaviour in the further course of hepatocarcinogenesis
- Regulation and expression of four cytochrome cytochromes P-450 isoenzymes, NADPH-reductase, the glutathione transferases B and C and microsomal epoxide hydrolase in preneoplastic and neoplastic lesions in rat liver
- Expression and inducibility of drug-metabolizing enzymes in preneoplastic and neoplastic lesions of rat liver during nitrosamine-induced hepatocarcinogenesis
- Alpha-fetoprotein: origin of a biological marker in rat liver under various experimental conditions
- Characterization, localization and regulation of a novel phenobarbital-inducible form of cytochrome P450-isoenzymes, NAPH P450-reductase, glutathione transferases and microsomal epoxide hydrolas
- Correlation of histology and alpha-1-fetoprotein resurgence in rat liver after experimental injury by galactosamine
- Hepatic progenitor cells, stem cells, and the biliary epithelium as transit compartment in models of liver injury
- Localization of alpha-1-fetoprotein and DNA-synthesis in liver cell populations during experimental hepatocarcinogenesis in rats
- Immunoperoxidase labelling of alpha-1-fetoprotein (AFP) in normal and regenerating livers of a low and a high AFP producing mouse strain
- Lectin-peroxidase conjugates in histopathology of gastrointestinal mucosa
- Cellular localization of lectin affinity in tissue sections of normal human duodenum
- Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat
- Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation
- Resin embedment of organs and postembedment localization of antigens by immunoperoxidase methods
- Ultrastructural immunoperoxidase cytochemistry
- Purification of mouse alpha-1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization
- Comparative study of the techniques for ultrastructural localization of antienzyme antibodies
- Comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates
- Localisation intracellulaire d’anticorps à l’aide de la glucose oxidase comme antigène et marqueur
- Immunohistochemical studies on human gastric mucosa. Procedures for routine demonstration of gastric proteins by immunoenzyme techniques
- Glucose oxidase as an antigen marker for light and electron microscopic studies
- Diaminobenzidine cytochemistry in cryoultramicrotomy for the detection of peroxidases
- Cross-linked albumin as supporting matrix in ultrathin cryo microtomy
- Immunoperoxidase technique in cryoultramicrotomy for the detection of simian adenovirus in cell nuclei
- Correlation of cell division and specific protein production during the course of lymphoid cell differentiation
- Lymphocyte differentiation and antibody synthesis in the secondary immune response of peroxidase stimulated lymph nodes of rat
- Cellular differentiation and antibody localization during the primary immune response in peroxidase stimulated lymph nodes of rat
- Immunofluorescent histological demonstration of the antigenic gastric mucosal esterase VIA
in the superficial epithelium of human fundic gastric mucosa - Species specificity of the human gastric mucosa esterase VIA. I. Comparative studies on
gastric mucosa extracts of rabbit, guinea pig, rat and men
Photo gallery: immunohistology
Microscopic-histological detection of biochemically defined cell structures with monoclonal and polyclonal antibodies to detect defined substances in tissue preparations. Similarly, this applies also to other structures bound to special matrix to detect molecular binding properties. According to this scheme, qualitative and quantitative ligand assays with variable detection methods can be developed. Both direct and indirect forms of detection are possible.
- Principles and technical approaches for the detection of molecular staining in immunohistology and other ligand binding assays
- Expression of AFP in studies of hepatic regeneration and hepatocarcinogenesis
- Cellular detection of AFP in normal liver
- Serum AFP levels after acute injury and during hepatocarcinogenesis
- AFP expression by hepatocytes and biliary epithelial cells (oval-cell proliferation) during liver regeneration and chemical hepatocarcinogenesis
- AFP resurgence and some histological lesions observed in late stages of chemical carcinogenesis and in hepatoma development
- Localization of organ-type antigens in normal and diseased tissues
- Lymphocyte differentiation and detection of anti-enzyme antibody synthesis in lymph node cells after peroxidase (HRP) immunization
- Lectins as specific ligands for the study of glycoconjugates in cell research and histopathology
Photo gallery: methods and reagents
Material and laboratory equipment for the preparation of antigens, antibodies and antibody-enzyme conjugates. Processing of tissue samples for microscopy.
- Reagents for immunohistology
- Tissue preparation for immuno-enzyme studies
- Working area and technical equipment
References
Publications on microscopy, techniques in histology and relevant fields of work.