Histological technique

Electron microscopy Histochemistry Immunohistochemistry

Basics of electron microscopy

Electron microscope: The beam of electrons interacts with atoms in the tissue or atoms of heavy metals bound to the tissue (lead, osmium, uranium)

Shorter wavelength theoretical resolution 0,2 nm biological samples not better than 1.5 nm

Components of transmission electron microscope

The electron gun

Processing of tissue for EM

Fixation
Embedding Sectioning Contrasting

Equipment and accesories for EM technique

HISTOCHEMISTRY
Histochemical methods
- are based on the specific chemical reactions, producing insoluble colored or electron dense reaction product - are used for detection of various chemical substances or expression of enzyme activity in tissue sections DNA Feulgens reaction RNA methylene or toluidine blue (control test with RNA-ase) LIPIDS Sudan dyes, Oil red (fat) Fast luxol blue MBS (phospholipids myelin sheath) POLYSACCHARIDES PAS reaction: glycogen, mucopolysaccharides (GAG), glycoproteins, glycolipids Alcian blue: acid mucopolysaccharides (mucus, heparin granules)

Polysaccharides
Polysaccharides in human organism occur as: glycogen (hepatocytes, cardiomyocytes), mucopolysaccharides/glycoaminglycans (in the mucus, ground substance of the extracellular matrix), glycoproteins, glycolipids. They can be demonstrated by PAS reaction. This histochemical method is based on the ability of Schiffs reagent (bleached basic fuchsin) to react with aldehyde groups to give distinctive purple (red, magenta) colour. Aldehyde groups of carbohydrates are revealed by oxidation with periodic acid.

Glycogen can be stained with Bests carmine (red colour).

Alcian blue is a special dye for staining acid mucopolysaccharides (GAG) in mucous and goblet cells.

Alcian Blue

PAS

PAS

Oil Red

Brain, Luxol blue + nuclear red White matter (myelin) - blue

Doc. MUDr. Vernerov

Detection of the activity of alkaline phosphatase

brush border of the absorptive epithelium of proximal tubules in kidney (T-5)

Azocoupling method
Incubation solution: alfa naphtol phosphate, Fast Red TR, buffer pH 9

Principle of azocoupling method

Enzyme splits substrate into phosphate and naphtol. Released naphtol is coupled with diazonium salt to form an insoluble colored azo-dye

Alkaline phosphatase azocoupling reaction - kidney

Alkaline phosphatase azocoupling reaction - kidney

Non-specific esterase kidney

T- 4, gallbladder, PAS reaction

Mucus and reticular fibers - purple

Gallbladder, alcian blue+nuclear red

Mucus is stained blue, nuclei red

Photomicrograph: doc. Jirkovsk

Collection of HIEM

IMMUNOHISTOCHEMISTRY
Methods based on the specifity of the reaction between ANTIGEN and ANTIBODY are used for detection of specific proteins and certain macromolecules. Antibodies are labelled by coupling with: Fluorescent compound (examination in fluorescence
microscope)

Enzyme: peroxidase, alkaline phosphatase (detection by

histochemical method, examination in light microscope) Gold particles (examination in electron microscope)

Direct method: labelled antibody binds to antigen Indirect methods: non-labelled antibody (primary) is bound to antigen. Secondary labelled antibody binds to primary antibody. This method is more sensitive.

Immunoglobulins - products of plasma cells IgA, IgD, IgE, IgG, IgM

IgG

IgM

Visualization of antibody binding to antigens imunofluorescence

Three step immunohistochemical reactions

Immunoperoxidase reaction

Alkaline phosphatase as an enzymatic label

Immunofluorescent detection