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IMMUNOFLUORESCENCE STAINING TECHNIQUE

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Presentation on theme: "IMMUNOFLUORESCENCE STAINING TECHNIQUE"— Presentation transcript:

1 IMMUNOFLUORESCENCE STAINING TECHNIQUE
by Sri Widyarti Disampaikan pada WORKSHOP IMMUNOFLUORESCENCE STAINING di LSIH Oktober 2019

2 Analysis based on Ag-Ab interaction
Western blotting / immunoblotting ELISA (enzyme-linked immunosorbent assay) Immunohistokimia / immunositokimia Immunofluorescence / IF (microscopic) Flowcytometry analysis (specific-cell counting)

3 Ag-Ab recognition fluorophore

4 higher than that of IHC > WB
METHODS WB ELISA IHC/ICC IF / Flowcyto STEPS complex; long cycle simple; short cycle simple; short cycle SENSITIVITY high; specificity simple but with high false-positive lower than that of WB higher than that of IHC > WB CROSS RX yes and easy to distinguish yes yes and hard to distinguish DATA qualitative quanitative positioning Positioning / Number of target cells SAMPLE MW Separated-proteins/ Membrane Homogenate / enzyme panel Sectiioning tissue / glass slide glass slide / cell suspension / tissue sectioning DYE RESULTS Chemical light, chromogenic product chromogenic product , fluorecescent Cromogenic Fluorescent OBSERAVTION TOOLS imaging Elisa reader Bright Field microscopy fluorescence microscopy / flowcytometer

5 ELISA WB IHC/ICC ELISA IF Flowcytometry WB only

6 What is immunofluorescence?
Is a technique for fluorescently labeling a specific biological target within a sample using an antibody. An antibody (an immunoglobulin), that binds specifically (but noncovalently) to another molecule (antigen or epitope). In immunofluorescence, the specificity of the fluorescent label comes from the specificity of the antibody for its antigen; the detection of the bound antibody is due to the fluorophore that is attached to the antibody

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8 HOW IS ANTIBODY RAISING

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10 Rabbit anti-X = rabbit IgG
Donkey anti-rabbit IgG

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15 Direct immunofluorescence
ADVANTAGES DISADVANTAGES Shorter sample staining time and a simpler workflow Offers the best solution for specific targeting Avoids any cross-reactivity between secondary antibodies Fluorophore signal relies on the finite number of fluorophores that can be attached to a single antibody Fewer colors may be available), more expensive

16 Indirect immunofluorescence
ADVANTAGES DISADVANTAGES Offers greater sensitivity relatively inexpensive, available in a wide spectrum of colors Potential for cross-reactivity Secondary antibody may react with endogenous immunoglobulin in tissue samples

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18 Tissue sections Frozen section (quick freezing in optimal cutting temperature (OCT) medium (IF-F) sectioned using a cryostat and allowed to air dry on the slide 10–15 minutes prior to fixation in the next step Embedding in paraffin (IHC-P). require deparaffination and antigen retrieval steps before incubation with antibodies.

19 Cells: Plating Conditions (IF-IC)
begin with seeding of cells on a support material compatible with fluorescence microscopy. Support formats include glass-bottom cell culture dishes, glass coverslips (kept in plastic culture dishes) prepared with polylysine and/or extracellular matrix components to support adherent cell culture, multiwell chambers mounted on glass slides that are compatible with microscopes.

20 Cells: Optimizing Cell Health and Density (IF-IC)
Ensure your cells remain healthy by regularly checking the culture media for pH changes and inspecting for signs of cell stress (such as multinucleated cells) at low magnification on a microscope. The confluence of the cells is appropriate for the cell type and target.

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22 Fixation and permeabilization
Key steps that can determine your experiment’s failure or success. The ideal fixative preserves a “life-like” snapshot while quickly stopping the degradative process of autolysis by crosslinking and inhibiting endogenous enzymes.

23 Choice of Fixative Aldehydes cross the plasma membrane and fix soluble proteins better than alcohols, but some targets can lose their antigenicity with aldehyde crosslinking Aldehydes react with and crosslink cellular proteins, stabilizing and hardening the sample. Formaldehida and glutaraldehyde are most commonly used. Recommends fixation with 4% formaldehyde.

24 Dehydrating/denaturing fixatives displace water around cellular macromolecules, resulting in their denaturation and precipitation in situ. Denaturation of the target protein may expose normally buried epitopes, making this approach advantageous for some antibodies. However, dehydrating fixatives are less suited for soluble targets and modification state-specific antibodies such as phospho-antibodies

25 Permeabilization If a crosslinking fixative is used, the plasma membrane will still be intact, making intracellular targets inaccessible to antibodies. Therefore, permeabilization should be performed after crosslinking fixation Triton X-100 permeabilization after fixation with the blocking step. Triton and other detergents such as NP-40, TWEEN, Saponin, Digitonin and DOTMAC remove different molecules from cellular membranes and create variable “pore” sizes to allow antibody access.

26 Methanol permeabilization improves performance of some antibodies.

27 Blocking Reduces background signal (caused by non-specific binding of primary and secondary antibodies to sites other than their intended target). Recommendation is 5% normal goat serum (or serum from the same species as the secondary antibody) in PBS + 0.3% Triton X-100.

28 Primary Antibody Dilution
It is important to use an antibody at its recommended dilution in order to obtain a high signal-to-noise ratio (S/N). If the antibody is applied at too low of a concentration, the fluorescence signal will be too dim to distinguish from background noise. An excessively high concentration will contribute to background staining, decreasing S/N. Diluted primary antibody is incubated overnight on 4 oC at dark-humid chamber.

29 Washing and Secondary Incubation
The antigen-binding region of an IF-approved antibody will have high affinity for its target epitope, but other regions of the antibody may still have lower affinity, non-specific interactions that could contribute to increased background. Washing the sample (3X for 5 min in PBS) helps to remove unbound antibody, prevent formation of primary-secondary immune complexes, and reduce unwanted background. Diluted secondary is applied for 1–2 hr at room temperature, protected from light.

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31 Counterstaining (Optional)
IF is ideal for investigating changes in subcellular localization because protein location can be analyzed in relation to labeled organelle markers, other proteins of interest, or other cellular structures. Common counterstains are phalloidins (Alexa Fluor 488 Phalloidin) to label actin filaments, PI to label DNA/nuclei, MitoTracker Red to label mitochondria.

32 Mount Sample Replacing the final PBS wash with a mounting agent such as Antifade Reagent provides several benefits: displacing water to prevent crosslinker depolymerization, curing (hardening) to stabilize the sample, more closely matching the refractive index of the optics (for oil immersion objective lenses), reduction of photobleaching by free radical scavenging.

33 Microscopic Observation

34 Light of a short wavelength generates light of a longer wavelength.
What is Fluorescence? Light of a short wavelength generates light of a longer wavelength. Illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence. Jablonski diagram Upon absorbing the excitation light, usually of short wavelengths, electrons may be raised to a higher energy and vibrational excited state excited electrons lose some energy & return to the lowest excited singlet state with Simultaneous emission of fluorescent light

35 Photobleaching Photobleaching - When a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification.

36 Photobleaching At low excitation intensities, photobleacing occurs but at lower rate. Bleaching is often photodynamic - involves light and oxygen. Singlet oxygen has a lifetime of ~1 µs and a diffusion coefficient ~10-5 cm2/s. Therefore, potential photodamage radius is ~50 nm.

37 Cell is stained with a dye
The dye is illuminated with filtered light at the absorbing wavelength The light emitted from the dye is viewed through a filter that allows only the emitted wavelength to be seen. The dye glows brightly against a dark background (only the emitted wavelength is allowed to reach the eyepieces or camera port of the microscope) EYEPIECES/CAMERA Second Barrier Filter Passes only Emitted Light LIGHT SOURCE Emitted Light Excitation Light Beam Splitting Mirror (Dichroic Mirror) First barrier Filter Passes only Excitation Wavelength (EXCITER FILTER) Objective Lens Cell

38 Properties of Fluorochrome
Excitation (nm) Emission (nm) Color DAPI 365 420 Blue Fluorescein 495 525 Green Hoechts 33258 360 470 R-phycocyanin 555, 618 634 Red B-phycoerythrin 545, 565 575 Orange, red R-phycoerythrin 480, 545, 565 578 Rhodamine 552 570 Texas red 596 620


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