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)