Immunofluorescence

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Immunofluorescence refers to use of antibodies in conjunction with fluorescent markers (also known as fluorophores). There are two approaches. In the direct (or primary) method the antibody against a specific antigen is conjugated with a fluorescent markers such as fluorescein (usually as fluorescein isothiocynate, FITC; green under UV light) and rhodamine (red under UV light). In the indirect (or secondary) method, the primary antibody is unlabelled and the fluorescent marker is attached to the secondary antibody. For instance, if the primary antibody is mouse IgG, the secondary antibody can be a fluorescein-conjugated rabbit anti-mouse antibody. The technique is typically performed on cell preparations or microtome-cut slices of tissue, placed on a glass slide and examined directly using a light microscope equipped with a ultraviolet light source.

Contents

Practicalities

Tissue transported in Michel's transport medium. This is not a fixative as such, but is a buffered isotonic solution that stabilises proteins until the antibody steps are carried out.

Applications

Renal

Dermatology

Immunofluorescence is a helpful adjunct in a number of bullous skin disorders. Several are characterised by autoimmune damage to specific cellular adhesion molecules either in the basement membrane or between keratinocytes. Using fluorophore-labelled antibodies against human IgG or IgA allows identification and localisation of these autoimmune immunoglobulins.

Immunofluorescence Patterns in Dermatological Conditions
Disease Clinical features Pathogenesis Histological appearence Immunofluorescence pattern Comments
Pemphigus foliaceus Superficial blisters, easily ruptured, but does not result in ulceration Auto-antibodies against Desmoglein 1 Subcorneal blisters Net-like pattern affecting predominantly the superficial epidermis More common in Brazil
Pemphigus vulgaris Affects skin and mucosa, especially face, scalp, trunk, axilla, groin and pressure points. Oral ulcers may precede skin blisters. Auto-antibodies against Desmoglein 3 (and sometimes 1 as well) Suprabasal blisters Net-like pattern of inter-cellular IgG and C3 in lower part or all of epidermis Represents 80% of pemphigus.
Bullous pemphigoid The blisters are harder to rupture as the roof consists of full thickness of epidermis, but when they do, it results in ulceration. Auto-antibodies against bullous pemphigoid antigen 2 (BPAG12), a component of the hemidesmosome that connects the basal layer to the basement membrane (dermo-epidermal junction). Sub-epidermal, non-acantholytic blisters + superficial (and sometimes deep) perivascular infiltrate consisting of neutrophils, eosinophils and lymphocytes. The basal layer can become vacuolated. Linear deposition of IgG and C3 along basement membrane
Dermatitis herpetiformis Urticarial, vesicular rash IgA antibodies produced against gliadin appear to cross-react with reticulin in the basement membrane In early lesions, neutrophils and fibrin develop at the tips of the dermal papillae. Basal vacuolisation occurs and the focal separation of the dermo-epidermal junction becomes confluent to form a subepidermal blister. Granular deposition of IgA along basement membrane, most pronounced at tips of dermal papillae Associated with coeliac disease
Systemic lupus erythematosus Auto-immune disease with multi-system effects Auto-antibodies against DNA, histones, nucleosomes, etc. Linear IgG band ('lupus-band test') and also positive signals from nuclei of keratinocytes (due to anti-nuclear antibodies). IgM, IgA, and C3 deposits may also be found in the basement membrane zone.
Henoch-Schonlein purpura IgA deposits in the walls of small dermal blood vessels
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