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Immunofluorescence
Introduction: 
 Immunofluorescence : Immunofluorescence is a 
powerful technique that utilizes fluorescent-labeled 
antibodies to detect specific target antigens.. 
 Fluorescein is a dye which emits greenish fluorescence 
under UV light. It can be tagged to immunoglobulin 
molecules. 
 This technique is sometimes used to make viral plaques 
more readily visible to the human eye. 
 Immunofluorescent labeled tissue sections are studied 
using a fluorescence microscope.
Examples Of Fluorescent Dyes 
Fluorescein Rhodamine
FLUORESCENCE MICROSCOPY
 There are two ways of doing IF staining 
 Direct immunofluorescence 
 Indirect immunofluorescence 
1. Direct immunofluorescence 
 It’s just a simple & a very common procedure in this 
regard. 
 Ag is fixed on the slide 
 Fluorescein labeled Ab’s are layered over it 
 Slide is washed to remove unattached Ab’s 
 Examined under UV light in an fluorescent microscope 
 The site where the Ab attaches to its specific Ag will 
show apple green fluorescence 
 Use: Direct detection of Pathogens or their Ag’s in tissues 
or in pathological samples.
Direct immunofluorescence
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.
 Advantage over direct IF 
Since several fluorescent anti-immunoglobulins 
can bind to each antibody present in the first 
layer, the fluorescence is brighter than the 
direct test. 
It is also more time-efficient since it is only one 
signal labelled reagent, the anti-immunoglobulin, 
is prepared during the lengthy 
conjugation process.
Confocal image to detect phosphorylated AKT (green) 
in cardiomyocytes infected with adenovirus
Immunofluorescence image of Cryptosporidium parvum oocysts
Concentrated groundwater methanotrophic bacteria on 0.2 m m 
filter labeled with fluorescent antibodies.
What Immunoflouroscence Does 
 Immunoflourescence is a Microscopic-based technique, 
used clinically to diagnose certain cutaneous diseases 
( e.g; Lyme Disease) by the detection of AG:AB 
Complexes. 
 Techniques including DIF, IDIF & Salt-split 
Skin are utilized depending on clinical scenario. 
 DIF is performed on patient’s skin using flourophore-labeled 
antibodies that directly bind to pathogenic 
autoantibody-antigen complexes in the skin.
What Immunoflouroscence Does: 
 IDIF techniques are used in Dermatology primarily to 
detect circulating pathogenic autoantibodies. 
LIMITATIONS 
 Fluorescence signals depend on the quality & 
Concentration of antibodies, proper handling of 
specimen & detection with appropriate secondary 
antibodies.
Immunofluorescence PPT

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Immunofluorescence PPT

  • 1.
  • 3. Introduction:  Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..  Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.  This technique is sometimes used to make viral plaques more readily visible to the human eye.  Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
  • 4. Examples Of Fluorescent Dyes Fluorescein Rhodamine
  • 6.  There are two ways of doing IF staining  Direct immunofluorescence  Indirect immunofluorescence 1. Direct immunofluorescence  It’s just a simple & a very common procedure in this regard.  Ag is fixed on the slide  Fluorescein labeled Ab’s are layered over it  Slide is washed to remove unattached Ab’s  Examined under UV light in an fluorescent microscope  The site where the Ab attaches to its specific Ag will show apple green fluorescence  Use: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples.
  • 8. 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.
  • 9.  Advantage over direct IF Since several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test. It is also more time-efficient since it is only one signal labelled reagent, the anti-immunoglobulin, is prepared during the lengthy conjugation process.
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  • 13. Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus
  • 14. Immunofluorescence image of Cryptosporidium parvum oocysts
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  • 17. Concentrated groundwater methanotrophic bacteria on 0.2 m m filter labeled with fluorescent antibodies.
  • 18. What Immunoflouroscence Does  Immunoflourescence is a Microscopic-based technique, used clinically to diagnose certain cutaneous diseases ( e.g; Lyme Disease) by the detection of AG:AB Complexes.  Techniques including DIF, IDIF & Salt-split Skin are utilized depending on clinical scenario.  DIF is performed on patient’s skin using flourophore-labeled antibodies that directly bind to pathogenic autoantibody-antigen complexes in the skin.
  • 19. What Immunoflouroscence Does:  IDIF techniques are used in Dermatology primarily to detect circulating pathogenic autoantibodies. LIMITATIONS  Fluorescence signals depend on the quality & Concentration of antibodies, proper handling of specimen & detection with appropriate secondary antibodies.