What is
histochemistry?
It is the study of qualitative identification & quantitative assessment of chemical groupings within cells and tissues.
Histochemical techniques are based on precise chemical rationales for their ability to identify or stain different biochemical substances.
Visualizing Chemicals and Enzymes in Tissue.
Based on chemical reactions between
cell components and stains.
The end products of the reaction are
permanent, colored precipitates that
can be viewed under the microscope.
There are stains specific to each
component of the cell, based on the
basic or acidic nature of the dye.
Applications
Origin- based on the chemical
substances
Progression
Microbial infections
Healing
Repair
Structure and chemical
composition of oral tissues
Epithelium and its derivatives
Connective tissue
Cells and fibers
Ground substance
Epithelium and its derivatives
Oral epithelium
Epithelial components of
tooth
Salivary glands
Cells and Fibers
Fibroblasts
Collagen (types I & III)
Reticular fibers
Elastic fibers
Connective tissue
Ground substances
Key role is adhesion and signaling events
Composition-Mixture of macromolecules
Proteoglycans
Cell surface associated macromolecules
Extra cellular macromolecules
Glycoproteins
Fibronectin- fibroblasts, smooth muscle, etc.,
Laminin- epithelial cells, basement membrane
Chondronectin- chondrocytes
Osteonectin- osteoblasts
Histochemical techniques
Fixation procedures
Chemical
Formaldehyde
Acrolein
Gluteraldehyde
Rossman’s fluid- glycogen, glycoproteins
Carnoy’s mixture- nucleic acid (Fuelgen reaction)
Paraformaldehyde- lipids
Non-chemical
Freeze drying- cytochrome oxidases
Freeze fracture- electron microscopy (3D picture
of cell membranes)
Histochemical study of teeth
and bone Simultaneous fixation and decalcification with
formaldehyde or gluteraldehyde and EDTA
(ethylenediaminetetraacetic acid) for light and
electron microscopic histochemistry.
Recently techniques have been developed for
sectioning freeze-dried, undecalcified tissues.
(eg: for studying DC).
Bone- deproteinization with conc. Hydrazine.
Specific histochemical methodsCarbohydrates- PAS
Proteoglycans- Toluidine blue, Azure A, Alcian
blue.
Proteins- Dinitrofluorobenzene, Ninhydrin or
Ferric ferricyanide
Lipids- Sudan dyes (Sudan black- phospholipids)
Enzymes (For eg-Phosphatases- Gomori
method)
Immunohistochemistry Immunohistochemistry takes advantage of
antigen-antibody affinity through its ability to
identify and localize proteins of interest via
detection with labelled conjugates (fluorescent
dyes).
Enzyme Histochemistry The techniques of enzyme histochemistry, which relate
structure and function, can be used to locate many enzymes,
including acid phosphatase, dehydrogenases, and
peroxidases.
Enzymes indicates what is happening in the tissue
Histochemistry of Oral hard
tissues
Carbohydrates(PAS)
Proteins (dinitrofluorobenzene, ninhydrin or
ferric ferricyanide)
Lipids(Sudan dyes)
Enzyme histochemistry
Alkaline phosphatase- absorption
Adenosine triphosphatase- pre absorptive
ameloblasts
Acid phosphatase- resorption
Esterase- matrix of bone and dentin
Aminopeptidase- human osteoclasts
Cytochrome oxidase- oxidation (osteoblasts,
osteoclasts)
Succinate dehydrogenase- osteoclasts
Histochemistry of oral soft tissues
Polysaccharides
Proteins
Lipids
Mucins(PAS for neutral mucins & alcian blue
for acid mucins)
Enzyme histochemistry
Alkaline phosphatase- endothelium, basement membranes
Acid phosphatase- degree of keratinization
Esterase- Gingiva(keratinized areas), salivary gland ducts, serous demilunes in sublingual gland, taste buds, mast cells.
Aminopeptidase
Succinate dehydrogenase and glucose-6-phosphate dehydrogenase
β-glucuronidase- basal layer
Cytochrome oxidase- crevicular epithelium
basal layer, ducts
of SG
Recent techniques In situ hybridization (identification
of gene or gene products)
Laser spectroscopy (inorganic
components of calcified tissue)
Confocal Laser scanning
microscope (3D picture)
Radioautographic techniques
(explains uptake of chemicals)