1352_Histological Technique

7/28/2019 1352_Histological Technique

1/30

Histological technique

Electron microscopyHistochemistry

Immunohistochemistry


2/30

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


3/30

Components of transmission electron microscope

The electron

gun


4/30


5/30


6/30


7/30

Processing of tissue for EM

Fixation

Embedding

Sectioning

Contrasting


8/30

Equipment and accesories for EM technique


9/30


10/30


11/30


12/30

HISTOCHEMISTRYHistochemical methods

- are based on the specif icchemical reactions, producinginso lub lecolored 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)


13/30

Polysaccharides

Polysaccharides in human organism occur as:glycogen(hepatocytes, cardiomyocytes),

mucopolysacchar ides/g lycoaminglycans(in the mucus, ground

substance of the extracellular matrix),

glycop roteins, glycol ip ids.

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.


14/30

Alcian Blue


15/30

PAS


16/30

PAS


17/30

Oil Red


18/30

Brain, Luxol blue + nuclear red

White matter (myelin) - blue

Doc. MUDr. Vernerov


19/30

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 naphtolphosphate, Fast Red TR, buffer

pH 9

Princ ip le of azocoupl ing methodEnzyme splits substrate into phosphate and naphtol. Released

naphtol is coupled with diazonium salt to form an inso lubleco lored azo-dye


20/30

Alkaline phosphatase azocoupling reaction - kidney


21/30

Alkaline phosphatase

azocoupling reaction - kidney


22/30

Non-specific esterase kidney


23/30

T- 4, gallbladder, PAS reaction Gallbladder, alcianblue+nuclear redMucus and reticular fibers - purple Mucus is stained blue, nuclei red

Photomicrograph: doc. Jirkovsk Collection of HIEM


24/30

IMMUNOHISTOCHEMISTRY

Methods based on the specifity of the reaction between

ANTIGEN and ANTIBODY are used for detection ofspecific proteins and certain macromolecules.

Antibodies are labelled by coupling with:

Fluorescent compound (examination in fluorescencemicroscope)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


25/30

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

IgG IgM


26/30

Visualization of antibody binding to antigens

imunofluorescence


27/30

Three step immunohistochemical reactions


28/30

Immunoperoxidase reaction


29/30

Alkaline phosphatase as an enzymatic label


30/30

Immunofluorescent detection

1352_Histological Technique

Documents

Transcript of 1352_Histological Technique