A brief outline of histochemistry

2. 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.

3. 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.

4. Applications
Origin- based on the chemical
substances
Progression
Microbial infections
Healing
Repair

5. Structure and chemical
composition of oral tissues
 Epithelium and its derivatives
 Connective tissue
 Cells and fibers
 Ground substance

6. Epithelium and its derivatives
 Oral epithelium
 Epithelial components of
tooth
 Salivary glands

7. Cells and Fibers
 Fibroblasts
 Collagen (types I & III)
 Reticular fibers
 Elastic fibers
Connective tissue

8. 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

9. 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)

10. 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.

11. Specific histochemical methods
Carbohydrates- 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)

12. 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).

14. 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

15. Histochemistry of Oral hard
tissues
Carbohydrates(PAS)
 Proteins (dinitrofluorobenzene, ninhydrin or
ferric ferricyanide)
Lipids(Sudan dyes)

16. 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

17. Histochemistry of oral soft tissues
 Polysaccharides
 Proteins
 Lipids
 Mucins(PAS for neutral mucins & alcian blue
for acid mucins)

18. 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

19. 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)