•Immunofluorescence is the labeling ofantibodies or antigens with fluorescentdyes.•Immunofluorescent labeled tissuesections are studied using a fluorescencemicroscope.• Fluorescein is a dye which emits greenishfluorescence under UV light. It can be taggedto immunoglobulin molecules.
There are two ways of doing IF staining◦ Direct immunofluorescence◦ Indirect immunofluorescence1. 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 fluorescentmicroscope The site where the Ab attaches to its specific Ag willshow apple green fluorescence Use: Direct detection of Pathogens or their Ag‟s intissues or in pathological samples
2. Indirect immunofluorescence: Indirect test is a double-layer technique The unlabelled antibody is applied directly tothe tissue substrate & Treated with a fluorochrome-conjugatedanti-immunoglobulin serum Advantage over direct IF◦ Because several fluorescent anti-immunoglobulins canbind to each antibody present in the first layer, thefluorescence is brighter than the direct test.
Principles of fluorescence∎ Light is an electromagnetic wave, characterised byfrequency 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 processExcitation coefficient ExcitationEmissionExtinction coefficient is the light capturing ability of thefluorophore molecule (high desired). A=Ecl.∎ Quantum yield is the efficiency of fluorescence ie.Ratio of emitted photons: absorbed photons (highdesired)∎ Stoke‟s Shift is the difference between energies of thethe absorbed photon and emitted photon.
∎ Implications-□ High Stoke‟s shift means greater heat produced□ Low Quantum yield means other non-radiativeprocesses are more likely to occur eg. collisionalquenching, internal conversion, intersystemcrossing□ Photobleaching results from reaction withoxygen in excited state. To avoid use low intensityexcitation, anti-fade agents, sensitive detectionsystem
Principle of FluorescenceFluorescence and phosphorescence are both types ofluminescence.When molecules with luminescent properties absorb light,they emit light of a different wavelength.• With fluorescence the emission of light occurs extremelyrapidly after the absorption of excitation light, whereaswith phosphorescence emission continues formilliseconds to minutes after the energy source has beenremoved.• Fluorescent materials give off light because of theiratomic structure.• Electrons are arranged in discrete energy levelssurrounding the atom‟s nucleus with each level having apredetermined amount of energy.
• When an electron absorbs the energy from aphoton 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-lifeof the excited state is generally less than 10 (8)seconds.• The electron loses a small amount of energy asheat and the remainder of the extra energy isgiven off in the form of a photon.• The emitted fluorescence has a lower energy thanthe absorbed light, so the wavelength of theemitted light is longer than that of the excitationlight (except in the case of multiphotonexcitation)
• A range of wavelengths of light can excite the electronsof a fluorochrome. For example, fluorescein will fluorescewhen hit by light with any wavelength between 450 nmand 520 nm.• However, the closer the excitation wavelength is to 495nm, the more fluorescence will be produced.• This optimal wavelength is called the excitation peak.• Similarly, the light produced by fluorochromes has arange of wavelengths.• The emission of light from fluorescein ranges from 490nm to 630 nm, and the emission peak is approximately515 nm.Since the phenomenon of fluorescence was first explainedby a British scientist, Sir George Stokes, in 1852, the shift inwavelength from short to long during fluorescence is called“Stokes shift” (Figure 4).
Figure 4. Excitation and emission spectrum of fluorescein.When fluorescein is excited at a wavelength other than itspeak excitation (470 nm in this example),the shape of the emission curve (darker green) remains thesame, but the relative intensity is reduced. The efficiency ofthe excitation at 470 nm is 45% of peak
Some fluorochromes have a small Stokes shift while otherfluorescent compounds have large Stokes shifts. Forexample, the fluorochrome fluorescein can be excited byblue-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 alarge Stokes shift. Thus, the light emitted is yellow-orange.In immunofluorescence, a single wavelength can be used toexcite several fluorochromes with differentStokes shifts and thereby produce a variety of fluorescentcolors asshown in Figure .ImmunofluorescenceExcitation spectrum: BlueEmission spectrum: GreenNucleus
The example in Figure shows a single wavelength at 488 nm(blue line) exciting three different fluorochromes identified bytheir absorption curves on the left of the figure (blue line). Eachfluorochrome is excited at a different efficiency and, therefore,the resulting emission will be at different intensities for equivalentfluorochrome concentrations. Knowing the excitation and emissionproperties of fluorescent compounds makes it possible to selectcombinations of fluorochromes that will work together. However, fora fluorochrome to be useful in a biological application it must attachto or be contained within a structure of biological significance.
Applications of Immunofluorescencein PathologySome practical applications of immunofluorescence in diagnosticpathology are:Analysis of antigens in fresh, frozen or fixed tissues; sub-cellularlocalization of antigens in tissue culture monolayers; observationof bacterial or parasitic specimens;Detection and localization of the presence or absence of specificDNA sequences on chromosomes; andDefining the spatial-temporal patterns of gene expression withincells/tissues.
• The most important application is in the field ofautoimmune.• It has wide application in demonstration of enzymes,hormones, plasma proteins, cells & its constituents.• In identifying viral, protozoal, bacterial and parasiticantigen.• IF methodes also have the potential to define antigen-antibody interactions at the subcellular level, such asdetection of antibodies against mitochondrias,microsomes and smooth muscle fibres.• As well as identifying small cell surface structures such asreceptors of lymphocytes.
After the biopsy,- The specimen can be placed on saline-saturated gauze & transported to thelaboratory in a sealed plastic container with ice- A pH of 7 to 7.2 should be maintainduring transport to reduce variablestaining- According to Carson (1997) extensivetime in medium can increaseAutofluorescence
Freezing of fresh unfixed tissue:- Tissue, that will not be frozen forseveral hours is best preserved using atransport medium such as ZEUS or MICHELmedium- Tissue should be fresh & freezingshould be done as rapid as possible usingoptimal cryostat temperature- Slow freezing can cause ice crystalformation which distorts tissuemorphology & antigen binding sites
The frequently used freezing techniques are asfollows;Liquid nitrogen (-190)Isopentane cooled by nitrogen (-150)Carbon dioxide „cardice‟ (-70)Carbon dioxide gas (-70)Aerosol spray (-50)Preferably 4um thick frozen sections areproduced using cryostat
Preparation of slides:- The slides should be prepared carefullyprior to procedure & can safely be stored foran indefinite period at room temp. in dustfree packingThe slides are first thoroughly cleaned as,Wash slides in detergent 30minWash slides in running tap water 30 minRinse slides in distilled water 2-5 min
Wash slides in 95% alcohol 3-5 minAir dry 10 min„Gelatin Formaldehyde‟(1% Gelatin 5ml +2% Formaldehyde 5ml)is used to avoid section floating off duringstainingCoat slides with this adhesive & allow to dry at 37cfor one hour before picking up sections
- Is used to detect Autoantibodies thatare bound to the patient‟s tissue- First the human immunoglobulins areinoculated in a goat which creates theantibodies directed against theseimmunoglobulins
- The antibodies are produced in the goatin response to human immunoglobulins- Then these are harvested from the goat& tagged with fluorescein
- A frozen section of patient‟s tissue isplaced on the slide & this is incubated withfluorescein - conjugated goat antihumanantibodies- These antibodies bind to the tissue at anysite where human immunoglobulin is present- The excess antibody suspension iswashed off & the section is viewed withfluorescence microscope
Staining technique:Slide preparation:Unfixed cryostat section, air dried, 4 microns thickMethod:1) Circle the location of the tissue on the backof the slide using a permanent pen; this willassist with identifying the tissue following staining
2) Place air dried slides in Tris buffer pH 7.6for 5 minutes3) Tap of and quickly remove excess buffer4) Place FITC-labeled antibody or negativeserum at its previously validatedconcentration on to the tissue section for 30minutes5) Rinse with Tris buffer to remove excessantibody
6) Rinse with deionizer water7) Coverslip using an aqueous mountingmedium8) Seal the edge of the coverglass with clearfingernail polish or permanent mounting medium9) Place slides in a slide tray10) Store the slides in a cool dark place untilreview
- Is a semiquantative procedure in which adouble immunolabeling is carried out toevaluate, the presence & titer of circulatingantibodies or to specifically localizeantigen in the skin- First a frozen section of tissue that issimilar to human oral mucosa (monkeyesophagus) is placed on the slide & incubatedwith the patient‟s serum
- If there are Autoantibodies directedagainst epithelial attachment structures inthe patient serum- Then they will attached to homologousstructure on the monkey esophagus- The excess serum is washed off
- Fluorescein conjugate goat antihumanantibody is incubated with section- The excess is washed off & the sectionis examined under fluorescent microscopeto detect the presence ofAutoantibodies that might have been inserum.
Staining technique: (Weller & coons 1954)Slide preparation:Unfixed cryostat section, air dried, 4 micronsthickMethod:1) Circle the location of the tissue on theback of the slide using a permanent pen;this will assist with identifying the tissuefollowing staining
2) Place air dried slides in Tris buffer pH 7.6for 5 minutes3) Tap of and quickly remove excess buffer4) Apply unlabeled antibody at itspreviously validated concentration on thetissue section for 30 Minutes5) Rinse using Tris buffer pH 7.6 for 5 minutes
6) Tap off & quickly remove excess buffer7) Apply avidin D for 15 minutes8) Rinse using Tris buffer pH 7.6 for 5minutes9) Tap off & quickly remove excess buffer
10) Apply d-biotin to tissue for 15 minutes11) Rinse using Tris buffer pH 7.6 for 5minutes12) Tap off & quickly remove excess buffer13) Apply biotinylated horse anti-mouseantibody for25 minutes14) Rinse using Tris buffer pH 7.6 for 5 minutes
15) Tap off & quickly remove excess buffer16) Apply fluorescein-streptavidin for 15minutes17) Rinse using Tris buffer pH 7.6 for 5minutes18) Rinse using deionized water for 5minutes19) Coverslip using an aqueous mountingmedium
20) Seal the edge of the coverglass with clearfingernail polish or permanent mountingmedium21) Place slides in a slide tray22) Store the slides in a cool dark place untilreview
Common dyes: fluorescein, rhodamine Dyes chosen are excited by a certain lightwavelength, usually blue or green, and emitlight of a different wavelength in the visiblespectrum◦ Eg. Fluorescein emits green light◦ Eg. Rhodamine emits orange/red light By using selective filters in a fluorescencemicroscope only the light from the dye isdetected Available fluorescent labels now include red,blue, cyan or yellow fluorescent proteins
This can be used to detect the distribution ofany protein By attaching different dyes to differentantibodies the distribution of two or moremolecules can be determined in the same cellor tissue sample
Direct Immunofluorescence is used todemonstrate the presence ofimmunoglobulins ,predominantly IgG butsometimes in combination with C3, IgA &IgM, in the intercellular substance in eitherthe oral epithelium of the lesions or ofclinically normal epithelium adjacent to thelesions. This test is carried out by incubating a biopsyspecimen with a fluoroscein-conjugatedantiglobulin.
Indirect Immunofluorescence is accomplishedbasically by incubating normal animal orhuman mucosa with serum from the patientsuspected of having the disease and addingthe fluorescein-conjugated humanantiglobulin. A positive reaction in the tissue indicates thepresence of circulating immunoglobulinantibodies.
DIF testing is very reliable & sensitivediagnostic test for pemphigus vulgaris, inthat it demonstrates lacelike IgG in thesquamous intercellular/cell surface areas inupto 95% of cases, including early cases &those with very few lesions,& in upto100%cases with active disease. IIF shows circulating IgG autoantibody insquamous intercellular substance in 80%-90%of cases.
DIF testing of perilesional skin is positive invast majority of cases. Two pattern ofpemphigus antibody deposition have beendescribed. In most cases there is fullthickness squamous intercellular substancedeposition of IgG. Rarely IgG may be localisedonly to superficial portion of epidermis. IIF testing of serum reveals squamousintercellular substance deposition of IgG in80% to 90% of cases.
DIF testing of perilesional skin revealssquamous intercellular substance depositionof IgG in>70% of cases & granular depositionof IgM & IgG at dermal epidermal junction. IIF study using monkey esophagus assubstrate reveals squamous intercellularsubstance deposition of IgG in 80% of cases.Antinuclear antibodies are observed in 30%-80% of cases.
DIF testing reveals IgA deposition in squamousintercellular substance throughout the epidermiswith increased intensity in the upper layers in somecase of subcorneal pustular type. However somecase may have both IgG & IgA present & thus maymake a specific diagnosis difficult(i.e. pemphigusvulgaris vs. IgA pmphigus). IIF results are positive in <50% of cases. InSPD(subcorneal pustular dermatosis) type IgAautoantibodies have been shown to recognizedesmocollin 1. In IEN(intraepidermalneutrohilic)type dermatosis the autoantigensremain to be identified; the antibodies have beenvariously characterised as reacting to desmoglein 1or desmoglein 3 in a subset of patients.
Incubation of normal or patient skin in 1mol/LNaCl results in split of epidermis in the laminalucida. Pemphigoid antibodies bind solely to thelower aspect of basal keratinocytes(the blister roof)in 80% of cases; in about 20% of cases antibodiesbind to both lower basal keratinocytes(the roof)andthe superior aspect of the dermis(the blister floor). When direct salt split skin technique is used inpemphigoid IgG is present on the roof or on theroof & the floor. Localization to only dermal base is characteristic ofEBA (epidermolysis bullosa aquisita) & p-200pemphigoid.
DIF testing of perilesional skin has shownlinear C3 deposition at the dermal epidermaljunction in virtually 100% of cases & IgG in65% to 95%. IIF studies reveal circulating anti-basementmembrane zone IgG antibodies in 70% to80%. Similarly deposited IgA & IgM areobserved in about 25% of cases.
Mucous membrane pemphigoid:- Autoimmune disease with autoantibodiesdirected against antigens BP180 (BPAg2) & epilligrin(laminin-5)BM appears to detach with the epitheliumfrom underlying connective tissue- DIF shows Continuous linear band mainly ofIgG & C3 at the BM zone, roof pattern of staining- IDIF shows Positive in only 5% of cases
DIF studies reveal linear IgG & C3 in lesional& perilesional skin in approximately 80% ofcases. Occassionaly IgA & IgM are alsopresent. IIF testing of serum shows circulatingantibodies more readily demonstrated whensalt-split human skin is used as substrate, inwhich IgG may be localised only to the roofor, as in antiepiligrin subgroup, to the base ofthe induced seperation.
Systemic lupus erythematosus:- DIF shows Shaggy, particulate deposits ofIgM, IgG or C3 at BM zone & Positive lupusband test- IDIF shows 95% - anti-nuclear antibodies,double- stranded DNA antibodies, Sm proteinantibodies which are very specific
Lichen planus:- DIF shows absence of immunoglobulindeposition in the basal lamina- The fluorescent pattern followsbasement membrane & fibrin depositionprobably accounts for the eosinophilicthickening of this region on routinestaining
- The fluorescence projects downwards in tosub mucosal in to an „icicle‟ or „stalactite‟appearance- Using IDF a subpopulation of patients hasbeen shown to possess circulating antibodiesagainst cytoplasm of basal keratinocytes- This phenomenon is more prevalent withlichenoid drug reactions than amongidiopathic drug reactions
DIF reveals IgA along the basementmembrane zone in perilesional skin in 100%cases. In lamina lucida type of LAD, IgAantibodies bind to the epidermal side of salt-split skin, whereas in sublamina densa type,such as IgA-mediated EBA, IgA antibodiesbind to the dermal side of salt- split skin.
Examination of perilesional skin using DIF revealslinear deposition of complement at the basementmembrane zone in vast majority of cases. IgG is by far most common immunoglobulinfound , but IgA & IgM may be present as well. IIF reveals circulting anti-basement membranezone antibodies in upto 50% The use of salt- split skin technique lead toappropriate diagnosis in most cases. Theantibodies in EBA have specificity for the globularcarboxyl terminus of typeVII collagen & aredeposited beneath the lamina densa. Therefore insalt-split skin studies IgG is on the floor & not onthe roof of the split.
In all reported cases, IgG & C3 are deposited atthe epidermal basement membrane zone. Thepattern was linear in more than 50% & wasreferred to as “granular bandlike” inapproxymately 25%.IgM &IgA were fluorescent inapproximately 50% to 60% of cases, respectively. IIF study of serum rarely reveals circulating anti-squamous basement membrane zone antibodiesthat are detected against typeVII collagen. A salt- split skin preparation using patient serumreveals localisation to the split floor, as in EBA
Presence of granular deposits of IgA within thedermal papillae in both lesional & nonlesionalskin. Fibrillary IgA deposits may also be present. Circulating IgA antibodies that react againstreticulin, smooth muscle endomyceum, thedietary antigen gluten, bovine serum albumin,B-lactoglobin may be present. Using monkey or pig gut as a substrate, IIF hasbeen used to detect antiendomysial antibodies,which are present in 52% to 100% patient.
In many patients with EM, deposits of IgM &C3 are found in the walls of the superficialdermal vessels. Granular deposits of C3, IgM& fibrinogen may also be present along thedermal-epidermal junction. Nonspecific deposition of immune reactantsin apoptic & necrotic keratinocytes may beobserved in other disorders with apoptosis,lymphocyte satellite necrosis,& epidermalnecrosis.
In general, IF results are often said to benegative. However, nonspecific granularbasement membrane zone staining of C3alone or in combination with IgM may befound.
1) It has high sensitivity making possiblethe demonstration of substances in very lowconcentrations or in particles below theresolution of transmitted-lightmicroscope.2) It can define the antigen-antibodyinteractions at sub cellular level, such asdetection of antibodies againstmitochondria, microsomes & smoothmuscle fibers.
3) It is useful in identifying small cellsurface structures such as receptors onlymphocytes.4) It has the ability to identify the exactsite of antigen-antibody reaction in tissue.5) It can identify the multiple antigenspresent on a single cell at a time, hence itis very useful in Research.
1) The stained section cant be reused, requiresfresh section every time.2) It is very technique sensitive, require afluorescent microscope.3) The ultraviolet lamp used in the fluorescentmicroscope has very short life span , so there isfrequent need to change the lamp, also thelamp is too costly
4) Ultraviolet light used in immunofluorescencetechnique is harmful to eyes.5) The observed immunofluorescence has to bestored in the form of photographs, as thefluorescence diminishes with time.6) It is not useful in electron microscope.7) False positive results can occur, as somesubstance shows auto fluorescence.
- Immunofluorescence is the adjunctivemethod for diagnosis of some diseases &premier method for diagnosis of autoimmune& mucocutaneous diseases.- For using this technique, completeknowledge of immunology on the molecularlevel must be understood- Fluorescent microscope-Types: direct & indirect
Class textbook (Human Molecular Genetics 3) www.antibodystation.com/immunofluorescence-microscopy/ National University of Singapore websitewww.med.nus.edu.sg/path/services/immunofluor.htm BD Biosciences websitewww.bdbiosciences.com Eversole Skin Histopathology