3. •Immunofluorescence is the labeling of
antibodies or antigens with fluorescent
dyes.
•Immunofluorescent labeled tissue
sections are studied using a fluorescence
microscope.
• Fluorescein is a dye which emits greenish
fluorescence under UV light. It can be tagged
to immunoglobulin molecules.

4. FLUORESCENCE MICROSCOPY

5.  There are two ways of doing IF staining
◦ Direct immunofluorescence
◦ Indirect immunofluorescence
1. Direct immunofluorescence
 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

7. 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
◦ Because several fluorescent anti-immunoglobulins can
bind to each antibody present in the first layer, the
fluorescence is brighter than the direct test.

9. Principles of fluorescence
∎ Light is an electromagnetic wave, characterised by
frequency and wavelength
∎ The electromagentic spectrum: 300m - 3 x 10 -12 m
∎ UV and visible light: 300nm – 750nm
∎ Fluorescence is the result of a 3 stage process
Excitation coefficient Excitation
Emission
Extinction coefficient is the light capturing ability of the
fluorophore molecule (high desired). A=Ecl.
∎ Quantum yield is the efficiency of fluorescence ie.
Ratio of emitted photons: absorbed photons (high
desired)
∎ Stoke‟s Shift is the difference between energies of the
the absorbed photon and emitted photon.

10. ∎ Implications-
□ High Stoke‟s shift means greater heat produced
□ Low Quantum yield means other non-radiative
processes are more likely to occur eg. collisional
quenching, internal conversion, intersystem
crossing
□ Photobleaching results from reaction with
oxygen in excited state. To avoid use low intensity
excitation, anti-fade agents, sensitive detection
system

11. Principle of Fluorescence
Fluorescence and phosphorescence are both types of
luminescence.
When molecules with luminescent properties absorb light,
they emit light of a different wavelength.
• With fluorescence the emission of light occurs extremely
rapidly after the absorption of excitation light, whereas
with phosphorescence emission continues for
milliseconds to minutes after the energy source has been
removed.
• Fluorescent materials give off light because of their
atomic structure.
• Electrons are arranged in discrete energy levels
surrounding the atom‟s nucleus with each level having a
predetermined amount of energy.

12. • When an electron absorbs the energy from a
photon of light (Figure 3) it becomes “excited”
and jumps to a higher, less stable energy level.
• The excited state does not last long. The half-life
of the excited state is generally less than 10 (8)
seconds.
• The electron loses a small amount of energy as
heat and the remainder of the extra energy is
given off in the form of a photon.
• The emitted fluorescence has a lower energy than
the absorbed light, so the wavelength of the
emitted light is longer than that of the excitation
light (except in the case of multiphoton
excitation)

13. Figure 3

14. • A range of wavelengths of light can excite the electrons
of a fluorochrome. For example, fluorescein will fluoresce
when hit by light with any wavelength between 450 nm
and 520 nm.
• However, the closer the excitation wavelength is to 495
nm, the more fluorescence will be produced.
• This optimal wavelength is called the excitation peak.
• Similarly, the light produced by fluorochromes has a
range of wavelengths.
• The emission of light from fluorescein ranges from 490
nm to 630 nm, and the emission peak is approximately
515 nm.
Since the phenomenon of fluorescence was first explained
by a British scientist, Sir George Stokes, in 1852, the shift in
wavelength from short to long during fluorescence is called
“Stokes shift” (Figure 4).

15. Figure 4. Excitation and emission spectrum of fluorescein.
When fluorescein is excited at a wavelength other than its
peak excitation (470 nm in this example),
the shape of the emission curve (darker green) remains the
same, but the relative intensity is reduced. The efficiency of
the excitation at 470 nm is 45% of peak

16. Some fluorochromes have a small Stokes shift while other
fluorescent compounds have large Stokes shifts. For
example, the fluorochrome fluorescein can be excited by
blue-green light, and its Stokes shift is only about 20 nm,
which means that the light emitted is green.
This contrasts with another fluorochrome, phycoerythrin,
which also can be excited by blue-green light, but has a
large Stokes shift. Thus, the light emitted is yellow-orange.
In immunofluorescence, a single wavelength can be used to
excite several fluorochromes with different
Stokes shifts and thereby produce a variety of fluorescent
colors as
shown in Figure .
Immunofluorescence
Excitation spectrum: Blue
Emission spectrum: Green
Nucleus

17. The example in Figure shows a single wavelength at 488 nm
(blue line) exciting three different fluorochromes identified by
their absorption curves on the left of the figure (blue line). Each
fluorochrome is excited at a different efficiency and, therefore,
the resulting emission will be at different intensities for equivalent
fluorochrome concentrations. Knowing the excitation and emission
properties of fluorescent compounds makes it possible to select
combinations of fluorochromes that will work together. However, for
a fluorochrome to be useful in a biological application it must attach
to or be contained within a structure of biological significance.

18. Applications of Immunofluorescence
in Pathology
Some practical applications of immunofluorescence in diagnostic
pathology are:
Analysis of antigens in fresh, frozen or fixed tissues; sub-cellular
localization of antigens in tissue culture monolayers; observation
of bacterial or parasitic specimens;
Detection and localization of the presence or absence of specific
DNA sequences on chromosomes; and
Defining the spatial-temporal patterns of gene expression within
cells/tissues.

19. • The most important application is in the field of
autoimmune.
• It has wide application in demonstration of enzymes,
hormones, plasma proteins, cells & its constituents.
• In identifying viral, protozoal, bacterial and parasitic
antigen.
• IF methodes also have the potential to define antigen-
antibody interactions at the subcellular level, such as
detection of antibodies against mitochondrias,
microsomes and smooth muscle fibres.
• As well as identifying small cell surface structures such as
receptors of lymphocytes.

21. After the biopsy,
- The specimen can be placed on saline-
saturated gauze & transported to the
laboratory in a sealed plastic container with ice
- A pH of 7 to 7.2 should be maintain
during transport to reduce variable
staining
- According to Carson (1997) extensive
time in medium can increase
Autofluorescence

22. Freezing of fresh unfixed tissue:
- Tissue, that will not be frozen for
several hours is best preserved using a
transport medium such as ZEUS or MICHEL
medium
- Tissue should be fresh & freezing
should be done as rapid as possible using
optimal cryostat temperature
- Slow freezing can cause ice crystal
formation which distorts tissue
morphology & antigen binding sites

23. The frequently used freezing techniques are as
follows;
Liquid nitrogen (-190)
Isopentane cooled by nitrogen (-150)
Carbon dioxide „cardice‟ (-70)
Carbon dioxide gas (-70)
Aerosol spray (-50)
Preferably 4um thick frozen sections are
produced using cryostat

24. Preparation of slides:
- The slides should be prepared carefully
prior to procedure & can safely be stored for
an indefinite period at room temp. in dust
free packing
The slides are first thoroughly cleaned as,
Wash slides in detergent 30
min
Wash slides in running tap water 30 min
Rinse slides in distilled water 2-5 min

25. Wash slides in 95% alcohol 3-5 min
Air dry 10 min
„Gelatin Formaldehyde‟
(1% Gelatin 5ml +2% Formaldehyde 5ml)
is used to avoid section floating off during
staining
Coat slides with this adhesive & allow to dry at 37c
for one hour before picking up sections

27. - Is used to detect Autoantibodies that
are bound to the patient‟s tissue
- First the human immunoglobulins are
inoculated in a goat which creates the
antibodies directed against these
immunoglobulins

28. - The antibodies are produced in the goat
in response to human immunoglobulins
- Then these are harvested from the goat
& tagged with fluorescein

29. - A frozen section of patient‟s tissue is
placed on the slide & this is incubated with
fluorescein - conjugated goat antihuman
antibodies
- These antibodies bind to the tissue at any
site where human immunoglobulin is present
- The excess antibody suspension is
washed off & the section is viewed with
fluorescence microscope

31. Staining technique:
Slide preparation:
Unfixed cryostat section, air dried, 4 microns thick
Method:
1) Circle the location of the tissue on the back
of the slide using a permanent pen; this will
assist with identifying the tissue following staining

32. 2) Place air dried slides in Tris buffer pH 7.6
for 5 minutes
3) Tap of and quickly remove excess buffer
4) Place FITC-labeled antibody or negative
serum at its previously validated
concentration on to the tissue section for 30
minutes
5) Rinse with Tris buffer to remove excess
antibody

33. 6) Rinse with deionizer water
7) Coverslip using an aqueous mounting
medium
8) Seal the edge of the coverglass with clear
fingernail polish or permanent mounting medium
9) Place slides in a slide tray
10) Store the slides in a cool dark place until
review

34. - Is a semiquantative procedure in which a
double immunolabeling is carried out to
evaluate, the presence & titer of circulating
antibodies or to specifically localize
antigen in the skin
- First a frozen section of tissue that is
similar to human oral mucosa (monkey
esophagus) is placed on the slide & incubated
with the patient‟s serum

35. - If there are Autoantibodies directed
against epithelial attachment structures in
the patient serum
- Then they will attached to homologous
structure on the monkey esophagus
- The excess serum is washed off

36. - Fluorescein conjugate goat antihuman
antibody is incubated with section
- The excess is washed off & the section
is examined under fluorescent microscope
to detect the presence of
Autoantibodies that might have been in
serum.

38. Staining technique: (Weller & coons 1954)
Slide preparation:
Unfixed cryostat section, air dried, 4 microns
thick
Method:
1) Circle the location of the tissue on the
back of the slide using a permanent pen;
this will assist with identifying the tissue
following staining

39. 2) Place air dried slides in Tris buffer pH 7.6
for 5 minutes
3) Tap of and quickly remove excess buffer
4) Apply unlabeled antibody at its
previously validated concentration on the
tissue section for 30 Minutes
5) Rinse using Tris buffer pH 7.6 for 5 minutes

40. 6) Tap off & quickly remove excess buffer
7) Apply avidin D for 15 minutes
8) Rinse using Tris buffer pH 7.6 for 5
minutes
9) Tap off & quickly remove excess buffer

41. 10) Apply d-biotin to tissue for 15 minutes
11) Rinse using Tris buffer pH 7.6 for 5
minutes
12) Tap off & quickly remove excess buffer
13) Apply biotinylated horse anti-mouse
antibody for25 minutes
14) Rinse using Tris buffer pH 7.6 for 5 minutes

42. 15) Tap off & quickly remove excess buffer
16) Apply fluorescein-streptavidin for 15
minutes
17) Rinse using Tris buffer pH 7.6 for 5
minutes
18) Rinse using deionized water for 5
minutes
19) Coverslip using an aqueous mounting
medium

43. 20) Seal the edge of the coverglass with clear
fingernail polish or permanent mounting
medium
21) Place slides in a slide tray
22) Store the slides in a cool dark place until
review

44.  Common dyes: fluorescein, rhodamine
 Dyes chosen are excited by a certain light
wavelength, usually blue or green, and emit
light of a different wavelength in the visible
spectrum
◦ Eg. Fluorescein emits green light
◦ Eg. Rhodamine emits orange/red light
 By using selective filters in a fluorescence
microscope only the light from the dye is
detected
 Available fluorescent labels now include red,
blue, cyan or yellow fluorescent proteins

45.  This can be used to detect the distribution of
any protein
 By attaching different dyes to different
antibodies the distribution of two or more
molecules can be determined in the same cell
or tissue sample

48. CLASSIFICATION:
1. Subcorneal split-
 Bullous impitigo
 Pemphigus foliacious
 Pemphigus erythematosus
 Subcorneal pustular dermatoses
 Staphylococcal scalded skin syndrome

49. 2. Spinous layer split-
 Friction blister
 Hailey hailey disease
 Ig A pemphigus
 Epidermolytic hyperkeratosis
3. Suprabasal split -
 Pemphigus vulgaris
 Dariers disease
 Grovers disease

50. 4. Subepidermal basement membrane zone
destruction-
(a) Lamina dura
 Epidermolysis bullosa
 Bullous pemphigoid
 Cicatritial pemphigoid
 Dermatitis herpatiformis
(b)Sublamina densa
 Epidermolysis bullosa dystrophica & acquistica
 Lineal IgA bullous dermatitis
 Bullous SLE

51. 5. Subepidermal split-
 Epidermolysis bullosa simplex
 Erythema multiforme
 Oral lichen planus.

52.  Direct Immunofluorescence is used to
demonstrate the presence of
immunoglobulins ,predominantly IgG but
sometimes in combination with C3, IgA &
IgM, in the intercellular substance in either
the oral epithelium of the lesions or of
clinically normal epithelium adjacent to the
lesions.
 This test is carried out by incubating a biopsy
specimen with a fluoroscein-conjugated
antiglobulin.

53.  Indirect Immunofluorescence is accomplished
basically by incubating normal animal or
human mucosa with serum from the patient
suspected of having the disease and adding
the fluorescein-conjugated human
antiglobulin.
 A positive reaction in the tissue indicates the
presence of circulating immunoglobulin
antibodies.

55.  DIF testing is very reliable & sensitive
diagnostic test for pemphigus vulgaris, in
that it demonstrates lacelike IgG in the
squamous intercellular/cell surface areas in
upto 95% of cases, including early cases &
those with very few lesions,& in upto100%
cases with active disease.
 IIF shows circulating IgG autoantibody in
squamous intercellular substance in 80%-
90%of cases.

57.  DIF testing of perilesional skin is positive in
vast majority of cases. Two pattern of
pemphigus antibody deposition have been
described. In most cases there is full
thickness squamous intercellular substance
deposition of IgG. Rarely IgG may be localised
only to superficial portion of epidermis.
 IIF testing of serum reveals squamous
intercellular substance deposition of IgG in
80% to 90% of cases.

58.  DIF testing of perilesional skin reveals
squamous intercellular substance deposition
of IgG in>70% of cases & granular deposition
of IgM & IgG at dermal epidermal junction.
 IIF study using monkey esophagus as
substrate reveals squamous intercellular
substance deposition of IgG in 80% of cases.
Antinuclear antibodies are observed in 30%-
80% of cases.

59.  DIF testing reveals IgA deposition in squamous
intercellular substance throughout the epidermis
with increased intensity in the upper layers in some
case of subcorneal pustular type. However some
case may have both IgG & IgA present & thus may
make a specific diagnosis difficult(i.e. pemphigus
vulgaris vs. IgA pmphigus).
 IIF results are positive in <50% of cases. In
SPD(subcorneal pustular dermatosis) type IgA
autoantibodies have been shown to recognize
desmocollin 1. In IEN(intraepidermal
neutrohilic)type dermatosis the autoantigens
remain to be identified; the antibodies have been
variously characterised as reacting to desmoglein 1
or desmoglein 3 in a subset of patients.

60.  Incubation of normal or patient skin in 1mol/L
NaCl results in split of epidermis in the lamina
lucida. Pemphigoid antibodies bind solely to the
lower aspect of basal keratinocytes(the blister roof)
in 80% of cases; in about 20% of cases antibodies
bind to both lower basal keratinocytes(the roof)and
the superior aspect of the dermis(the blister floor).
 When direct salt split skin technique is used in
pemphigoid IgG is present on the roof or on the
roof & the floor.
 Localization to only dermal base is characteristic of
EBA (epidermolysis bullosa aquisita) & p-200
pemphigoid.

61.  DIF testing of perilesional skin has shown
linear C3 deposition at the dermal epidermal
junction in virtually 100% of cases & IgG in
65% to 95%.
 IIF studies reveal circulating anti-basement
membrane zone IgG antibodies in 70% to
80%. Similarly deposited IgA & IgM are
observed in about 25% of cases.

64. Mucous membrane pemphigoid:
- Autoimmune disease with autoantibodies
directed against antigens BP180 (BPAg2) & epilligrin
(laminin-5)
BM appears to detach with the epithelium
from underlying connective tissue
- DIF shows Continuous linear band mainly of
IgG & C3 at the BM zone, roof pattern of staining
- IDIF shows Positive in only 5% of cases

66.  DIF studies reveal linear IgG & C3 in lesional
& perilesional skin in approximately 80% of
cases. Occassionaly IgA & IgM are also
present.
 IIF testing of serum shows circulating
antibodies more readily demonstrated when
salt-split human skin is used as substrate, in
which IgG may be localised only to the roof
or, as in antiepiligrin subgroup, to the base of
the induced seperation.

68. Systemic lupus erythematosus:
- DIF shows Shaggy, particulate deposits of
IgM, IgG or C3 at BM zone & Positive lupus
band test
- IDIF shows 95% - anti-nuclear antibodies
,double- stranded DNA antibodies, Sm protein
antibodies which are very specific

70. Lichen planus:
- DIF shows absence of immunoglobulin
deposition in the basal lamina
- The fluorescent pattern follows
basement membrane & fibrin deposition
probably accounts for the eosinophilic
thickening of this region on routine
staining

72. - The fluorescence projects downwards in to
sub mucosal in to an „icicle‟ or „stalactite‟
appearance
- Using IDF a subpopulation of patients has
been shown to possess circulating antibodies
against cytoplasm of basal keratinocytes
- This phenomenon is more prevalent with
lichenoid drug reactions than among
idiopathic drug reactions

73.  DIF reveals IgA along the basement
membrane zone in perilesional skin in 100%
cases. In lamina lucida type of LAD, IgA
antibodies bind to the epidermal side of salt-
split skin, whereas in sublamina densa type,
such as IgA-mediated EBA, IgA antibodies
bind to the dermal side of salt- split skin.

74.  Examination of perilesional skin using DIF reveals
linear deposition of complement at the basement
membrane zone in vast majority of cases.
 IgG is by far most common immunoglobulin
found , but IgA & IgM may be present as well.
 IIF reveals circulting anti-basement membrane
zone antibodies in upto 50%
 The use of salt- split skin technique lead to
appropriate diagnosis in most cases. The
antibodies in EBA have specificity for the globular
carboxyl terminus of typeVII collagen & are
deposited beneath the lamina densa. Therefore in
salt-split skin studies IgG is on the floor & not on
the roof of the split.

76.  In all reported cases, IgG & C3 are deposited at
the epidermal basement membrane zone. The
pattern was linear in more than 50% & was
referred to as “granular bandlike” in
approxymately 25%.IgM &IgA were fluorescent in
approximately 50% to 60% of cases, respectively.
 IIF study of serum rarely reveals circulating anti-
squamous basement membrane zone antibodies
that are detected against typeVII collagen.
 A salt- split skin preparation using patient serum
reveals localisation to the split floor, as in EBA

77.  Presence of granular deposits of IgA within the
dermal papillae in both lesional & nonlesional
skin. Fibrillary IgA deposits may also be present.
 Circulating IgA antibodies that react against
reticulin, smooth muscle endomyceum, the
dietary antigen gluten, bovine serum albumin,
B-lactoglobin may be present.
 Using monkey or pig gut as a substrate, IIF has
been used to detect antiendomysial antibodies
,which are present in 52% to 100% patient.

78.  In many patients with EM, deposits of IgM &
C3 are found in the walls of the superficial
dermal vessels. Granular deposits of C3, IgM
& fibrinogen may also be present along the
dermal-epidermal junction.
 Nonspecific deposition of immune reactants
in apoptic & necrotic keratinocytes may be
observed in other disorders with apoptosis,
lymphocyte satellite necrosis,& epidermal
necrosis.

79.  In general, IF results are often said to be
negative. However, nonspecific granular
basement membrane zone staining of C3
alone or in combination with IgM may be
found.

81. 1) It has high sensitivity making possible
the demonstration of substances in very low
concentrations or in particles below the
resolution of transmitted-light
microscope.
2) It can define the antigen-antibody
interactions at sub cellular level, such as
detection of antibodies against
mitochondria, microsomes & smooth
muscle fibers.

82. 3) It is useful in identifying small cell
surface structures such as receptors on
lymphocytes.
4) It has the ability to identify the exact
site of antigen-antibody reaction in tissue.
5) It can identify the multiple antigens
present on a single cell at a time, hence it
is very useful in Research.

84. 1) The stained section cant be reused, requires
fresh section every time.
2) It is very technique sensitive, require a
fluorescent microscope.
3) The ultraviolet lamp used in the fluorescent
microscope has very short life span , so there is
frequent need to change the lamp, also the
lamp is too costly

85. 4) Ultraviolet light used in immunofluorescence
technique is harmful to eyes.
5) The observed immunofluorescence has to be
stored in the form of photographs, as the
fluorescence diminishes with time.
6) It is not useful in electron microscope.
7) False positive results can occur, as some
substance shows auto fluorescence.

87. - Immunofluorescence is the adjunctive
method for diagnosis of some diseases &
premier method for diagnosis of autoimmune
& mucocutaneous diseases.
- For using this technique, complete
knowledge of immunology on the molecular
level must be understood
- Fluorescent microscope
-Types: direct & indirect

88.  Class textbook (Human Molecular Genetics 3)
 www.antibodystation.com/immunofluorescen
ce-microscopy/
 National University of Singapore website
www.med.nus.edu.sg/path/services/immunof
luor.htm
 BD Biosciences website
www.bdbiosciences.com
 Eversole Skin Histopathology

89. THANK YOU