1. IMMUNOFLUORESCENCE STAINING TECHNIQUEby
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

8. HOW IS ANTIBODY RAISING

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

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

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.

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.

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