2. Oral cavity is comprised of both hard and soft tissues.
These tissues possess different biochemical substances in them.
Various types of chemicals can stain these substances present in
the tissues.
3. Histochemistry identifies the position and distribution of
substances like carbohydrates, lipids, nucleic acids, proteins,
enzymes, pigments and minerals present in a tissue
4. Primarily oral tissues are composed of
• Epithelium
• Connective tissue
• Salivary glands
5. The chemical constituents involved in these tissues are
• Proteoglycans
• Glycoproteins
• Protein
• Lipids
• Enzymes
6. Derivatives of epithelium
Mucin or mucoid content in salivary glands of glandular
epithelium
Strongly sulphated mucin,
Weakly sulphated mucin,
Carboxylated mucin,
Sulphated sialomucin and
Neutral mucins.
13. Cells and fibres
Fibroblasts are the predominant cells
They elaborate collagen, reticular fibres and elastic fibres
They help in wound healing, repair and development.
Collagen and reticular fibres stain positively for glycoproteins
with silver stain and PAS.
14. Cells and fibres
Elastic fibres are stained by
Aldehyde fuchsin,
Resorcin fuchsin and
Orcein dye.
15. Histochemical techniques
Fixation
• Maintains tissue in a lifelike condition and helps to study them
exactly in the same way they were in the human body
• Prevents autolysis and putrefaction
• Decrease the various changes that happen to the cytoplasmic
and extracellular macromolecules. which includes enzymes,
structural protein- carbohydrate complexes, lipids and nucleic
acids as well.
16. Fixation and fixatives
• 10% formaldehyde
• Rossman’s fluid composed of Formaldehyde, Picric acid and
acetic acid
• Carnoy’s mixture composed of ethyl alcohol, acetic acid and
chloroform
• Acrolein and glutaraldehyde are aldehydes used for fixation
17. Fixation and Fixatives
• Imidazole buffered Osmium tetrachlroride is a post fixative
and helps in localizing lipid rich in unsaturated fatty acids.
• Post fixation or secondary fixation is done on lipid rich tissues
and freeze dried tissues.
• Uranyl acetate , a post fixative is used to preserve the
phospholipid membrane and dehydration with acetone
minimizes the extraction of phospholipids.
19. • Freeze fracture and freeze etching technique gives excellent
three dimensional images of the surface of various cell
membranes.
• For studying the histology of bone and teeth, Formaldehyde or
glutaraldehyde and EDTA are used.
20. SPECIFIC HISTOCHEMICAL
METHODS
These can be classified into three groups
• Glycogen, glycoprotein and proteoglycan methods
• Protein and lipid methods
• Enzyme methods
21. Glycogen, glycoprotein and
proteoglycan methods
Periodic Acid Schiff (PAS) technique is the best known technique
for detection of carbohydrates
Periodic acid
oxidises
glycol groups
Schiff’s reagent
Aldehydes Reddish purple
Product
23. Proteoglycans
Proteoglycans are demonstrated by Thiazine dyes like Toluidine
blue, Azure A and Alcian blue
Localization of proteoglycans are done by cationic dyes like
• Ruthium red
• Silver tetraphenyl porphine sulfonate
• Bismuth Nitrate and
• Cuprolinic blue
24. Proteins
Reagents used for protein Histochemistry
• Dinitroflurobenzene
• Ninhydrin
• Ferric ferricyanide
25. Lipids
Frozen or freeze dried sections are needed to study
Histochemistry of lipids.
Histochemistry of Lipids
• Total Lipids - Sudan dyes
• Phospholipids - Sudan black
• Ultrahistochemical reaction of Phospholipid - Iodopalatine
• Phosphatidylserine and Sphingomyelin - Malachite
Green aldehyde
26. Enzymes
Enzymes like phosphatases are demonstrated by popular
methods like
• Gomori method
• Coupling Azo dye technique
• Glucose -6- phosphatase enzyme is demonstrated by
Wachstein and Meisel’s method.
27. Immunohistochemistry
• IHC combines histological, Immunological and biochemical
techniques for the identification of specific tissue components
by means of specific antigen/ antibody reaction tagged with a
visible label.
• IHC makes it possible to visualize the distribution and
localization of specific cellular components within a cell or
time.
29. Histochemistry of Oral Hard
Tissues
Carbohydrates
The ground substance of teeth and bone is best visualized in PAS
stain.
When enamel is vizualized with PAS, enamel lamellae stain but
enamel matrix is non-reactive.
Interglobular dentin, dentinogenesis imperfecta and odontome,
have intense reactivity with PAS stain.
30. Protein
Seen in Proteins of dentin undergoing decay and in
developmental stages of tooth formation
DINITROFLUOROBENZENE (DNFB) and NINHYDRIN SCHIFF are of
interest in studying teeth and bone
31. Lipids
There is a low lipid content in mature dentin but enamel rod
sheath and odontoblastic process have high phospholipid
content, hence Sudanophilic
32. Enzymes
Enzymatic activity associated with teeth and bones are
demonstrated in the following
• Alkaline phosphatase
• Acid phosphatase
• Amino peptidase
• Cytochrome oxidase
• Succinic dehydrogenase
33. Alkaline phosphatase
Associated with mineralization
Activity is seen
• Endosteum,
• Periosteum,
• Osteocytes,
• Stratum Intermedium,
• Odontoblasts adjoining Korff’s fibres and
• Ground substance of developing molars and incissors.
34. Acid phosphatase
• This enzyme is localised in lysosomes of cell.
• Acid phosphatase activity is seen in osteoclasts of bone and
Odontoclasts of resorbing bone.
35. Histochemistry of Oral Soft
Tissues
Carbohydrates
• The commonly used stain for glycogen, proteoglycans and
glycoprotein is Periodic acid Shiff (PAS) reaction.
• Salivary mucins are identified by Mucicarmine stain.
36. • Frozen sections are used to study lipid histochemistry.
Commonly used stains are
• Sudan black
• Oil Red.
37. Proteins
In Oral epithelium, certain areas show keratinization normally.
Pathologically it can occur anywhere in mouth. Degree of
keratinization can be analysed by Ferric Ferricyanide method.
38. Enzymes
In human gingiva, the capillary endothelium of lamina propria
shows Alkaline Phosphatase activity and so does basement
membranes of salivary gland acini.
40. Enzymes Reactivity noted
Esterases
Superficial layers of gingiva
Salivary gland ducts
Serous demilunes of
sublingual gland
Aminopeptidase
Basal layers of epithelium &
connective Tissue of Gingiva
Salivary gland ducts
Glucoronidase
Basal layers of oral epithelium
Cytochrome oxidase
Basal layer of free and
attached gingiva
Crevicular epithelium and
epithelial attachment
Chronic gingivitis
Salivary duct system
Succinate dehydrogenase
Basal cells of gingival
epithelium
Salivary ducts
Malignant lesion of oral
mucosa
41. Angiogenic factor
• 67KD protein, an angiogenic factor was found in macrophages
of inflamed gingival tissue and inflamed tissue of rheumatoid
origin.
• Based on pore size and permeability, differentiation is made
between connective tissue components like muscle and
collagen
43. Verhoeff Van Gieson stain showing
elastin stained in black and
collagen in red
44. Advanced techniques
Immunofluorescense
Detection of biomolecules, antigens, cytoplasmic fibres and
nuclei
Visualization of antigen and specific proteins in tissue sections by
binding antibody chemically conjugated with a fluorescent dye
Antibodies are labelled with a compound that makes them glow
an apple green colour under ultraviolet light
46. In situ hybridization
• Hybridisation technique used in detection and localization of
specific DNA and RNA sequences in cells and whole tissue
• Binding of a nucleotide probe to a specific target sequence of
DNA or RNA
• The hybridisation is detected by fluorescent dyes in FISH or
with enzymatic probes like in immunoperoxidase or with
radio-isotope molecules like P32, H3 or S35
47. Polymerase chain reaction
• A technique sensitive diagnostic material, which aids in
amplification of a specified segment of DNA and make billions
of copies of the same
• PCR has undergone multiple advancements like pathogens
and novel genes identification and nucleotide sequence
quantification
49. ELISA
• Enzyme linked Immunosorbent Assay procedure helps in
detecting and quantifying substances which could be a
peptide, protein, antibody or a hormone
• The procedure utilizes an enzyme that is conjugated and binds
with an antigen- antibody complex
• A colored reaction measurable end product results when a
substrate is added
51. Confocal microscopy
Technique for improving optical resolution and contrast of a
micrograph by using a spatial pinhole blocking out of focus light
in image formation
It helps in viewing 3 dimensional images with better contrast and
less haziness
This allows examination of fluorescent labelled thick specimens
without physical sectioning.
52. Flow cytometry
• It is technique sensitive and can detect immunophenotpe of
cells in a specimen with thousand cells.
• It also can analyse the DNA content of cells
53. Radioautographic techniques
• This technique enables in identifying the chemical substances
uptake by metabolic pathways of different tissues in different
regions of cytoplasm.
54. Clinical considerations
• Need for Histochemistry to diagnose pathologies arising in
oral cavity
• histochemical stains for identifying tumors based on its origin
and content
• Tumor markers aid in accurate identification of tumor based
on its tissue of origin.
55. Clinical considerations
• PCR, FISH help in identifying chromosomal aberrations and
gene products
• ELISA helps in identifying the presence of an antigen in a
sample.
Microbial infections need special stains to identify
specific organisms, thereby paving way for
accurate diagnosis which helps in appropriate
treatment and also avoiding unnecessary antibiotic
intake by the patient.
