Histological technique III

1
Histological technique III

• Electron microscopy
• Histochemistry
• Immunohistochemistry

Group 1051 teacher Tomáš Kucera
2
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
Components of transmission electron microscope
The electron gun
4
(No Transcript)
5
(No Transcript)
6
(No Transcript)
7
Processing of tissue for EM

• Fixation
• Embedding
• Sectioning
• Contrasting

8
Equipment and accesories for EM technique
9
(No Transcript)
10
(No Transcript)
11
(No Transcript)
12

• 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)

13
Polysaccharides Polysaccharides in human
organism occur as glycogen (hepatocytes,
cardiomyocytes), mucopolysaccharides/glycoaminglyc
ans (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.
14
Alcian Blue
15
PAS
16
PAS
17
Oil Red
18
Brain, Luxol blue nuclear red White matter
(myelin) - blue
Doc. MUDr. Vernerová
19
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
20
Alkaline phosphatase azocoupling reaction -
kidney
21
Alkaline phosphatase azocoupling reaction -
kidney
22
Non-specific esterase kidney
23
T- 4, gallbladder, PAS reaction
Gallbladder, alcian bluenuclear red Mucus and
reticular fibers - purple Mucus is stained
blue, nuclei red
Photomicrograph doc. Jirkovská

Collection of ÚHIEM

24
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.

25
Immunoglobulins - products of plasma cells IgA,
IgD, IgE, IgG, IgM
IgG
IgM
26
Visualization of antibody binding to antigens
imunofluorescence
27
Three step immunohistochemical reactions
28
Immunoperoxidase reaction
29
Alkaline phosphatase as an enzymatic label
30
Immunofluorescent detection