IMMUNOFLUORESCENCE

Nashwa elsayed mohammedAssitant Lecturer

Microbiology & Immunology Departement

Faculty of medicine

By

FLUORESCENCE MICROSCOPE

COMPONENTS

Light source(xenon arc lamp or mercury vapor lamp)

Excitation filter

Dichoric mirror

Emission filter

Fluorescence microscopePrinciple:

Light source is U.V. mercury

vapor lamp

U.V. has a very short wave

length not visible to eye

Fluorescence microscope

U.V. is used to illuminate organism,

cells ,particles which have been

previously stained with fluorescent

dye called fluorochromes

These dyes dyes are capable of

transforming short wave length U.V.

light into longer wave length visible

by eye

Fluorescence microscope

The fluorescence which is given off by

the specimen passes through a

barrier filter located between objective

and eyed piece which ensures that all

only fluorescence wave length specific

for specimen reach eye piece

Short wave length from fluorescence

lamp passes through excitation

filter and is directed onto the

dichroic mirror located above

specimen

Dichroic mirror reflects shorter

wave length and allows longer wave

length to pass

Principle of Fluorescence Microcopy

Principle of Fluorescence Microcopy

Exciter filter

Principle of Fluorescence Microcopy

Exciter filter Barrier filter

Epi-fluorescence microscope

Transmitted light flurorescence

microscope

Common fluorescent dyes

fluorescein isothiocyanate (green)

Rhodamine (red)

Alexa flours

Dylight flours

Acridine Orange

Calcofluor white

The basic principle of immunofluorescence

To use a fluorescent compound (usually fluorescein) to detect the binding of antigenand antibody

The Ab is labelled with the fluorescent compound

Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurs

Types

Direct immunofluorescence

Indirect immunofluorescence

Avidin-Biotin Complex (ABC) Method

Direct immunofluorescence

Antigen is fixed on the slide Fluorescein labeled antibodies are layered

over it Slide is washed to remove unattached

antibodies Examined under UV light in an fluorescent

microscope The site where the antibodies attaches to its

specific Ag will show apple green fluorescence

2. Indirect immunofluorescence:

Indirect test is a double-layer technique

The unlabelled antibody is applied directly to the tissue substrate

Treated with a fluorochrome-conjugated anti-immunoglobulin serum

Avidin-Biotin Complex (ABC) Method ABC method is one of widely used technique

for immunhistochemical staining.

Avidin, a large glycoprotein, can be labeled with fluorescein and has a very high affinity for biotin.

Biotin, a low molecular weight vitamin, conjugated to antibodies.

Avidin-Biotin Complex (ABC) Method The technique involves three layers.

The first layer is unlabeled primary antibody.

The second layer is biotinylated secondary

antibody.

The third layer is a complex of avidin-biotin

FITC complex

Photobleaching

Fluorophores lose their ability to fluorescence as they are illuminated in a process called photobleaching

Can be controlled by: Reducing intensity of light exposure

Increasing concentration of fluorophores

Use more robust fluorophores (alexa flours or daylight flours)

Antifading agents as propyl gallate may be added to the glycerol mounting fluid

Immunofluorescense

Positive immunofluorescence test for

rabies virus antigen. (Source: CDC)

(Virology Laboratory, Yale-New

Haven Hospital)

Immunofluorescence image of Cryptosporidium parvum oocysts

Advantages and Disadvantages

Advantages

Result available quickly, usually within a few

hours.

Inexpensive

Able to detect a range of pathogens

Influence the decision to administer antibiotics

Benefit in early diagnosis and antiviral therapy in

immunocompromized

Advantages and Disadvantages

Potential Problems

Often very much reduced sensitivity compared to

cell culture

Requires good specimens.

Requires fluorescence microscope

Slide preparation are not permanent

Practical part

Cell spot preparation

1. Transfer specimen to a conical

centrifuge tube and centrifuge at

300 g for 10 minutes at 4ºC

2. Remove and discard the supernatant

3. Tap the tube gently to loosen the

cells, add PBS, and suspend the

cells by gently pipetting up and

down.

4. Repeat the centrifugation step.

5. Remove and discard the supernatant

6. Tap the tube and add enough PBS to

yield an opalescent cell suspension.

7.-Deliver 35 μl of the cell suspension in

the well of a Teflon-coated slide and see

under the microscope to ensure that the

cell concentration is adequate

8.-Air dry the slides completely at

room temperature.

9.-Fix the cells by immersing the slide

in acetone for 10 minutes at 4ºC.

10.-Remove the slides from acetone

and allow them to air dry completely

at room temperature.

Staining procedure

1. Add 25 μl of FITC-labelled

monoclonal antibody to each well

and incubate for 30 minutes at 37ºC

in a humid chamber.

2. Wash with PBS for 10 minutes.

Staining procedure

3. Dry and add buffered glycerin

4. Place a coverslip and press slightly to

avoid bubbles.

5. View under the fluorescence

microscope at 400 x.

Interpretation of results

Adenovirus negative (IF) Adenovirus positive (IF)