electron microscopy and histochemistry maňáková 2009
TRANSCRIPT
Electron microscopy and histochemistry
Maňáková
2009
Electron microscopy
Ernst Ruska with co-worker constructed first electron microscopy in Germany in 30th
He obtained the Nobel price for physics in 1986. 1st 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 Fe2+
Fe2+ (HCl) andpotassium ferrocyanide. Product of reaction isPrussian blue(Atlas No.1)
Detection of organic compounds
Carbohydrates: polysaccharides (glycogen)
glycoproteins and proteoglycans glycolipids
(PAS reaction – HIO4 + Schiff reagent)
PAS
Basic fuchsin andSodium metabisulphite=leucofuchsin,ie. Schiff reagentIt reacts with aldehydicgroup 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 brushborder (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 partVariable part is importantfor binding to epitop, constant
is typical for animal in whichantibody 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