Electron microscopy and histochemistry Maňáková 2009

Electron microscopy Ernst Ruska with co-worker constructed first electron microscopy in Germany in 30 th He obtained the Nobel price for physics in st EM was made by Siemens and Halske Resolution power TEM 0,2 nm SEM 10 – 15 nm Analytical electron microscopy can detect elements from 5 (boron)

Resolution power Resolution power TEM 0,2 nm SEM 10 – 15 nm Analytical electron microscopy can detect elements from 5 (boron)

Principle Source: cathode+anode shape of disc with aperture Wehnelt´s cylinder Condensor Objective Projective

Method of ultra-thin section Sampling Fixation (glutaraldehyde, paraformaldehyde and osmium oxide)‏ Embedding (epoxide, polyester and acrylate resins)‏ Polymeration Cutting - thickness 50-60nm Contrasting (osmium, uran, tungsten)‏ Observation

Grid

Method of negative staining Corpuscle is surrounded by electron-dense substance – phospho- tungsten acid or uranyl acetate = dense background, particles are light Used for detection of viruses

Cryofracture – freeze fracture Frozen tissues are fractured, coated by metal dust, observed in TEM Structure of membranes

T10 – Vein - FMA

T11 – Cerebrum -Toluidin Blue

T12 – Kidney -Toluidin blue

What is necessary to know? How the objects are prepared for observation in TEM ? What are semi-thin sections? How we can detect viruses in EM? Negative staining. Resolving power of SEM and TEM

Histochemistry It uses chemical and histochemical reaction for the detection of elements or compounds in situ in cells and tissues Histochemistry Catalytic histochemistry Affinity histochemistry

Detection of elements or compounds Elements: Hg, Pb, Fe, Ca, Zn and their salts Perls reaction –detection of Fe 2+ Fe 2+ (HCl) and potassium ferrocyanide. Product of reaction is Prussian blue (Atlas No.1)‏

Detection of organic compounds Carbohydrates: polysaccharides (glycogen) glycoproteins and proteoglycans glycolipids (PAS reaction – HIO 4 + Schiff reagent)

PAS Basic fuchsin and Sodium metabisulphite= leucofuchsin, ie. Schiff reagent It reacts with aldehydic group on sugars

PAS (Atlas No.2)‏

Detection of organic compounds Lipids (lipid soluble dyes)‏ Sudan dyes: Sudan black, Sudan IV, oil red

Detection of organic compounds DNA – Feulgen reaction (HCl + Schiff reagent)‏

Catalytic histochemistry It allow detection of enzymes (enzymatic activities) in tissues and cells Used for: Research: localization of enzymes in cell Diagnostic: celiac disease They serves as markers for visualization in affinity histochemistry

Principle 1. histochemical reaction Tissue with Enzyme + Substrate = Product 2. reaction –visualisation Coloured and insoluble compound arises from colourless product of first reaction

Conditions: To maintain the enzymatic activity and structure of tissue and cells, we use cryostat sections Fixation degreases or inhibits completely the enzymatic activity pH, temperature, substrate in abundance Activators and inhibitors For 200 enzymes are available special procedures - protocols

Catalytic histochemistry 1.reaction – condition should be similar to those in tissue 2. reaction – only few reactions exist for visualization, whole groups of enzymes are proceeded by the same reaction. More methods are available for only few enzymes (phosphatases)‏ We have to make probative slides before histochemical reaction. Sections are stained usually by methylene blue

Methods for visualization in catalytic histochemistry Precipitation Metal salt capture methods (Cobalt, Lead, cerium)‏ Diazonium salt methods Indigogenic methods Tetrazolium salt methods DAB- diaminobenzidine methods

Aminopeptidase M Visualization for all peptidases –diazonium salt method (Atlas No.4)‏

Alkaline phosphatase Localisation (where is it in the cell) Distribution (where it is in tissue)‏ Alcaline phosphatase is enzyme of brush border (Atlas No.3)‏

Na, K ATPase is an enzyme using the same substrate however it has different localization in cell, it is present in the baso- lateral labyrinth (Atlas No.5)‏

Affinity histochemistry Immunohistochemistry – detection of proteins (glycoproteins) by the binding of the specific antibody to the antigen Lectin histochemistry –detection of mono-, di-, tri-, i polysaccharides in the complex molecules by binding of lectins to the saccharides In situ hybridization – detection of specific sequence of nucleoids in DNA or m-RNA by the binding of complementary chain of probe

Markers Antibodies (lectins and probes, too) have to be visualized by markers: Fluorochromes (FITC, rhodamine) Biotin Enzymes (HRP, alP) – catalytic histochemistry is used for their visualization Colloidal gold particles Isotopic probes Ferritin, digoxigenin

Immunohistochemistry Antibody is composed from 2 heavy and 2 light chains. It has variable and constant part Variable part is important for binding to epitop, constant is typical for animal in which antibody was produced

Polyclonal a monoclonal antibodies Antibody binds to specific place on protein – epitop Antibodies– polyclonal monoclonal

Immunohistochemistry Direct reaction Ag + AB Indirect reaction Ag + AB1 + AB2

PAP reaction Peroxidase – Anti-peroxidase Signal amplification More molecules of enzyme mean much product and heavier signal

ABC reaction Biotin is marker of secondary antibody It reacts with avidin that is bound to the enzyme Signal is amplified

Cytokeratins (Atlas No.6)‏

Insulin (Atlas No.8)‏

Immunohistochemistry is used for : Diagnostic of tumores and other illnesses in pathology The most important antigens: Intermediate filaments, CD antigens, hormones, estrogen and progesteron receptor, melanoma antigens, S-100 protein, PSA (prostatic specific antigen), proliferation specific antigens: PCNA, p53 protein, KI-67 Research

Lectins Lectins are proteins or glycoproteins that agglutinate cells and/or precipitate complex carbohydrates. The binding is highly specific. Lectins are isolated from a wide variety of natural sources including plants, fungi, bacteria and vertebrates. Application: blood grouping mitogenic stimulation of cells (lymphocytes) histochemical studies

In situ hybridisation Detection of specific sequences DNA or m-RNA by binding of complementary probes, that must be labelled by marker -fluorochrome (FISH) or enzyme

What is necessary to know How we can detect basic components of tissue and cells? What we detect by Perls reaction, PAS, Feulgen reaction? Principle of catalytic histochemistry and its application Principle of immuno-histochemistry and its application. How we demonstrate proteins in tissue? Which markers are used in affinity histochemistry