Double Labeling Fluorescent Immunocytochemistry Protocol

Materials of Double Labeling Fluorescent ICC

Cells Plating and Treatment with Rapamycin and Chloroquine(See Note 1)

1. LN-229 human glioblastoma cells.
2. Tissue culture flasks, dishes, tubes, multi-well plates.
3. Glass coverslips, chamber slides with removable chamber (see Note 2).
4. Watchmaker's forceps/tweezers.
5. Incubator.
6. Light inverted microscope.
7. Phosphate buffered saline (PBS).
8. Sterile-filtered trypsin solution: 0.25% trypsin, 0.02% EDTA, PBS.
9. Cell culture medium: Dulbecco's Modified Eagle's Medium—low glucose (with 1000 mg/L glucose, L-glutamine, and sodium bicarbonate), 10% fetal bovine serum (FBS) or fetal calf serum (FCS), penicillin–streptomycin (stock concentration—10,000 units penicillin and 10 mg streptomycin/mL; use 100 μL/50mL of media [1:100 dilution]).
10. Cell culture medium (from item 9) with 100 nM rapamycin.
11. Cell culture medium (from item 9) with 100 μM chloroquine.
12. Cell culture medium (from item 9) with solvent used for dissolving rapamycin and chloroquine.

Labeling

1. Glass coverslips, glass slides.
2. PBS.
3. 10% neutral buffered formalin—NBF (see Note 3).
4. 0.25% Triton X-100 in PBS (see Note 4).
5. Glycine/Tris (optional—see Note 5).
6. Blocking solution of 1% BSA (bovine serum albumin) in PBST:1% BSA, 0.05% Tween 20, PBS (see Note 5).
7. Primary antibodies mixture: 1:400 dilution of mouse antiphospho-IRS-1 (Ser612) IgG antibody and 1:100 dilution of rabbit anti-LC3A/B IgG antibody in blocking solution (from item 6).
8. Secondary antibodies mixture: 1:500 dilution of goat anti-mouse IgG conjugated with Alexa Fluor 488 and 1:500 dilution of goat anti-rabbit IgG Alexa Fluor 568 in blocking solution (from item 6).
9. Petri dishes.
10. Parafilm.
11. Distilled water.
12. Marker.
13. Aluminum foil.
14. Plate shaker or rocker enabling gentle rocking of dishes/plates/chamber slides.
15. 1 μg/mL Hoechst 33342 in PBS—optional, if using mounting medium without DAPI.
16. Razor blade.
17. Mounting medium with DAPI.
18. Nail polish.
19. Paper towels.
20. Watchmaker's forceps/tweezers.
21. Fluorescence microscope.

Notes of Double Labeling Fluorescent ICC

1. All solutions, dishes, and equipment used for cell culture coming into contact with live cells must be sterile. Aseptic technique should be used.
2. You can choose different dishes to plate and stain cells. One of the options is using multi-well chamber slides with removable chamber that are dedicated for staining. They may be a good solution, for example, if you do not have enough cells or materials to use bigger dishes. Another method is to utilize glass coverslips placed in culture dishes, for example, multi-well culture plates. If you choose the second option, make sure that you have proper size glass coverslips, that will fit in the dish/ well of the multi-well plate and it will be possible to easily place them in and take them out of the dish/well with tweezers. You can also use a needle or needle with tip bent near the end to retrieve coverslips from the dishes/wells easier. Also remember that chamber slides in which you grow cells on a slide thickness piece of glass, may not be suitable for some applications and imaging methods.
3. 10% neutral buffered formalin (NBF) is used as fixing agent. You can also use freshly prepared 4% paraformaldehyde (PFA) in PBS. Formaldehyde is a crosslinking fixative widely used for cells and tissues. It can cause some undesired effects, for example, induce fluorescence or excessive crosslinking that may reduce signal (over-fixing of samples should be avoided to prevent false negative results). There is a wide choice of other fixing agents as well, for example, ice-cold methanol. Fixing agent should be chosen and optimized to preserve specific antigen that will be visualized and its distribution, as well as cells and organelles morphology. Some antibodies may not detect antigens processed with some fixatives, for example, formaldehyde or glutaraldehyde. Always check the protocols and application notes of antibodies for this information. Some fixing agents can also be used to permeabilize cells, for example, acetone, methanol.
4. Triton X-100 is used as permeabilizing agent. Depending on the detected antigen, other permeabilizing agents can be used, for example, saponins and digitonin are mild alternatives. Fixation and permeabilization protocols should be carefully considered as some fixation/permeabilization procedures may cause false subcellular localization.
5. BSA is used as blocking solution. Other blocking agents may be, for example, normal (nonimmune) serum from the same species that the secondary antibody was raised in, fetal calf serum (FCS)/fetal bovine serum (FBS), casein protein, nonfat dry milk, and gelatin. Glycine or tris may be added to blocking solution to quench unreacted formaldehyde and terminate cross-linking reaction. Formaldehyde quenching can abolish nonspecific attachment of antibodies preventing high background/ false positive staining.
6. Coverslips and slides should be prepared. Coverslips can be sterilized by autoclaving. Also using UV enabled laminar flow culture hood, soaking in 70% ethanol, washing in ethanol and flaming or combination of ethanol washing and UV are used to prepare coverslips for cell culture.
7. Some cultured cell types that can be easily grown on plastic, do not adhere well to glass surface. Cell attachment can be enhanced with surface coatings or other methods. They should be used carefully as they may, for example, change cell behavior or show autofluorescence, for example, collagen. Poly-L-lysine is widely used for many cell lines. Acid washing procedure of coverslips cleaning may also enhance cell attachment.
8. Cells can also be seeded in a bigger culture dish and transferred to multi-well plate when they reach desired confluency or after conducting planned experimental procedures.
9. When plating cells on coverslips placed in culture dishes ensure that the coverslips do not float in the medium but remain on the bottom of the dish. If coverslips are floating, push them down to the bottom using sterile pipette tip or tweezers.
10. In all steps use reagents in the amount covering cells. Do not let cells dry out throughout the whole procedure.
11. Selection of antibodies specific to the antigen of interest is critical for the staining. All available validation data, products citations, and user reviews should be taken into consideration. It is recommended to follow manufacturer's suggestions and try a range of dilutions for initial characterization. Binding specifically to the unique epitope on the target antigen is especially important, for example, when detecting various post-translational modifications or isoforms of a protein. Also, reactivity that refers to the species of the antigen detected by a given antibody is crucial. If the antibody was not tested to work against desired species, comparing the epitope sequence of the verified vs. unverified species can be helpful in predicting antibody reactivity. Monoclonal antibodies are often used as primary antibodies because of their ability to specifically bind to a single epitope of an antigen. Although, for example, for antigens that are present in low quantities—polyclonal antibodies could be beneficial. Because of binding to different epitopes, they could amplify the signal and ensure better detection. For this reason, they are also amplifying the signal used as secondary antibodies. For applications where no variability in antibodies is acceptable, recombinant antibodies are recommended. They are custom synthetic antibodies providing unlimited supply of identical batches over time, but at higher cost. Peptide antibodies can also show advantages. In indirect detection that requires the use of secondary antibody (against primary antibody) labeled with fluorochrome, the host of an antibody should be considered. It refers to the species of the animal used to produce the antibody. Each secondary antibody should be directed against the species of the corresponding primary antibody. When more than one primary antibody is used, primary antibodies raised in different species are recommended, if possible, to minimize secondary antibody cross-reactivity. Staining with antibodies raised in the same species requires a different protocol. A secondary antibody should be directed against, but not raised in, the same species as the primary antibody. It should also be directed against the class or subclass of the primary antibody. For polyclonal primary antibodies anti-IgG secondary antibody is usually recommended because most polyclonal antibodies are typically IgG class antibodies. In multiple labeling experiments, it is recommended to use all secondary antibodies derived from the same host species, if possible. If primary antibody raised in a closely related species has to be used, a secondary antibody preadsorbed against the related species can be considered. It decreases binding of a primary antibody raised in a closely related species but may decrease the signal. When staining cells with Fc receptor expression (e.g., macrophages, natural killer cells), it is recommended to use Fab or F(ab0)2 fragment secondary antibody. They eliminate nonspecific binding, as they do not have Fc fragment that could be bound to Fc receptor.
12. In multiple labeling when primary antibodies from different hosts are not available, use of different class (such as IgG, IgM) or subclass (IgG1, IgG2b, IgG3, etc.) specific primary and secondary antibodies may be beneficial. When using antibodies of different classes, it is important to remember about secondary antibody class specificity. Anti-Fc fragment antibodies and heavy chain specific antibodies are class specific. Antibodies that recognize light chains are not class specific and react with all classes of antibodies.
13. Appropriate controls should be performed in each experiment to eliminate the possibility of false-positive or false-negative results. In general controls can be divided into three groups: primary antibody controls—to show the specificity of primary antibody binding the antigen, secondary antibody controls—showing the labeling is specific to the primary antibody, and labeling/detection controls—to show the labeling is the result of label added during the procedure and not endogenous signals. They are also divided into positive and negative controls.
    a) Autofluorescence control—unstained samples, that were subject to all steps of the staining procedure except immunostaining (antibodies treatment) and counterstaining.
    b) Control without primary antibody; secondary antibody control—samples fixed, blocked, permeabilized and incubated with secondary antibody. When using multiple primary antibodies, controls should be also prepared without each primary antibody, but with all of the secondary antibodies, in order to verify if secondary antibodies are binding to the correct primary antibodies and to test if the secondary antibodies are binding to each other.
    c) Isotype control—cells incubated with nonimmune serum antibodies of the same isotype from the same species (or normal/preimmune serum) instead of primary antibody.
    d) Negative expression control—staining of cells lacking the expression of the antigen of interest (e.g., cells derived from knockout models, but one should be careful if the knockdown is only functional still expressing nonfunctional, mutated protein) or with gene-mediated silencing (shRNA/siRNA).
    e) Positive expression control—staining of cells known to express the target protein.
    f) Absorption control; preadsorption control/test—labeling with primary antibody blocked with a specific antigen, blocking peptide or protein (it may be the immunogen used for antibody production).
    g) Single-stained controls in multicolor experiments— should be prepared and examined with filter sets for all used fluorophores to verify the level of bleed-through.
14. When selecting secondary antibodies fluorophores, it is important to check the lasers and filter sets of the microscope that is available to ensure optimal excitation and detection. Next, it is crucial to select fluorophores with minimal spectral overlap to avoid bleed-through (also termed crossover, crosstalk or spillover, although there are some differences in terminology) artifacts that are important to avoid, especially in colocalization studies or quantitative measurements like FRET (Fo¨rster Resonance Energy Transfer). Fluorescence spectrum viewers available online may be helpful in planning that aspect of experiments. A good example is employing commonly used green fluorochrome together with the red one for detection of antigen that is much more abundant than the other one—false positive result can occur because of their spectral characteristics. It is advised to use red fluorophore for very strong signal and green for the weaker one. In multicolor studies, it is recommended to associate the brightness of a fluorochrome with antigen abundance. The brightest fluorophore should be used to detect the least abundant antigen and the dimmest one to detect the most abundant cellular targets. In general, fluorophores with green and red emission are brighter than those emitting in the blue and far-red portions of the spectrum. The brightness of fluorochromes can be checked by verification of extinction coefficient (measurement of probability to absorb a photon of light) and it is directly proportional to it. The higher extinction coefficient, the brighter the fluorochrome. There are some other fluorophores parameters that can be taken into consideration (e.g., quantum yield). Nevertheless, in multicolor experiments, single-stained control specimens should always be examined with filter sets for all used fluorophores to assure that the level of bleedthrough is minimal. It is also advisable to check photobleaching (also termed fading) properties of fluorochromes. In photobleaching fluorophore loses the ability to fluoresce because of photon-induced chemical damage and covalent modification. Conventional fluorochromes (FITC, PE) are often sensitive to photobleaching, while families of Alexa Fluor^® or DyLight fluorophores are more photostable. For fluorochromes prone to photobleaching antifade mounting medium can be used. Also, during microscope imaging, lowering the intensity or exposure time of the excitation light and using higher gain or pixel binning settings may help reduce fading. In the process of carefully planning the use of fluorophores, it is essential to test sample autofluorescence as well and prepare appropriate control not stained with fluorochromes.
15. Cells are usually counterstained to identify a particular cellular organelle or structure, and to easily identify individual cells. Most commonly used counterstain dyes are DAPI, Hoechst, propidium iodide for DNA staining and fluorescently labeled phalloidin (actin cytoskeleton staining) or wheat germ agglutinin (stains plasma membrane). Counterstaining can be performed as a separate step in the labeling procedure, but dyes can also be added to antibodies mixtures. There is also a selection of mounting media containing different dyes available. It is important to match their spectral properties with the fluorochromes labeling antibodies.
16. Different mounting media can be used—some of them harden and some remain liquid. They may contain antifading agents to protect samples from photobleaching.
17. Sealing with nail polish can be used to prevent the sample from drying out, prevent leak out of the mounting medium and to keep the coverslip in place. However, nail polish can be fluorescent itself and also cause other issues making it not suitable for some applications.