Fixation of special substancesPRESENTATION BY

LARAIB AYESHA

MLT 2ND YEAR

Fixation• In fields of histology,patholology and

cell biology fixation is a chemical process by which biological tissues are preserved from decay,thereby preventing from

• Autolysis• Putrefaction• Fixation terminates ongoing biochemical

reactions • It also increase the mechanical strenght or

stability of treated tissues.

Special substances • Which are to be fixed are following• Proteins• Lipids• Carbohydrates• Biogenic amines • Nucleic acids• Water and lipid soluble substances

Fixation of Proteins• Crosslinking fixatives “Aldehydes”• Crosslinking fixatives act by creating covalent chemical

bonds between proteins in tissue. • Most commonly used fixative in histology is the crosslinking

fixative formaldehyde (also named formalin). • Formaldehyde is the most commonly used fixative for amino

acid histochemistry.• Another popular aldehyde for fixation is glutaraldehyde.

• Glutaraldehyde fix proteins very rapidly. • Glutaraldehyde may not penetrate thicker tissue

specimens as effectively as formaldehyde. • Glutaraldehyde may offer a more rigid or tightly linked

fixation.

Fixation of lipids• In order to demonstrate lipids,frozen sections

are commonly used.• Baker’s formal calcium used to preserve

phospholipids.• For preservation of lipids in cryostat sections

fixative should contain• Mercuric chloride• Potassium dichromate• For this purpose we use the Elfman’s fluid.• Fixation is over 3 days at room temperature.

Frozen sections

Fixation of carbohydrates• Alcoholic fixatives are used for glycogen

fixation.

are• Rossman’s fluid or ethanol.• Fixatives for ultrasturctural preservation of

dextrans and glycogen is

• Simionescu’s fixative• It is used by making stock solutions of• Formaldehyde• Glutaraldehyde• Osmium tetraoxide• Lead citrate

Fixation of biogenic amines• Biogenic amines can be demonstrated in

tissues using a variety of techniques.• Chromaffin reaction

In this reaction potassium dichromate-formaldehyde is used.

• Formaldehyde vapour fixation

Formaldehyde fixation of the adrenal medulla produced fluorescence of cells which contained biogenic amines

• Freeze dried paraffin sections or carnoy fixed paraffin section

For histamines• Primary biogenic amines are well preserved

by Glutaraldehyde.

Fixation of nucleic acids• Fixatives choosen for nucleic acids must depend upon

subsequent methods which are to be employed in analysis of tissue.

• Formaldehyde –based fixatives (used but mostly avoided)

• Carnoy fixative – (most widely used) • Feulgen reaction • It is to be used then the hydrolysis time varies with the

fixative.

Fixation of water and lipid soluble substances• For accurate demonstration of water soluble

substances,non-aqueous fixatives are used.

For example, Urates in gout• Osmium tetroxide is a satisfactory fixative

For steriods.

Target Fixatives of choice

Fixatives of avoided

proteins Natural buffered formalin, paraldehyde

Osmium tetroxide

lipids Frozen sections Chemical fixative

Nucleic acid Alcoholic fixative Aldehyde fixative

Glycogen Alcoholic based fixatives Osmium tetroxide