Fluorescent Dyes

The steps of immunofluorescence staining experiment

Immunofluorescence staining is a commonly used biochemical examination method, which is often used for the localization of antigens or antibodies in histology, and can also be used for the quantitative detection of antigens or antibodies in body fluid samples. Many small partners encountered various problems when doing immunofluorescence staining experiments. In fact, it is not difficult to successfully complete an immunofluorescence staining experiment after mastering the principle, operation steps and precautions of immunofluorescence staining experiment.

1. What is immunofluorescence technology?
Immunofluorescence technique (Immunofluorescence technique), also known as fluorescent antibody technique, the main principle is to use the specific binding between antigen and antibody to display the target protein. Immunofluorescence technology not only has high specificity of antigen-antibody reaction, but also can clearly display its shape under fluorescence microscope, which is highly intuitive.

There are two types of immunofluorescence staining experiments, including direct immunofluorescence and indirect immunofluorescence.

​How to solve the strong background color of immunofluorescence staining? Explain the experimental operation and precautions in detail

Direct immunofluorescence is the earliest method. The basic principle is to use a known antibody to label fluorescein to become a specific fluorescent antibody. When staining, the antibody is directly dropped on the slide for incubation, so that it can directly bind to the antigen on the slide and can be directly detected under a fluorescence microscope. Observe and make judgments.

The basic principle of indirect immunofluorescence method is to use a specific antibody to bind to the antigen in the slice, and then use an indirect fluorescent antibody to combine with the previous antigen-antibody complex to form an antigen-antibody fluorescent complex. Under a fluorescence microscope, the detected antigen is determined by the luminescence of the complex.

The basic principle of both methods is the same. Immunofluorescence (IF) or cell imaging techniques use antibodies to label fluorescent dyes (also called fluorescein or fluorophores), such as fluorescein isothiocyanate (FITC), to specific target antigens. Antibodies chemically conjugated to fluorescein are widely used in IF experiments.

Direct immunofluorescence method is simple, specific, fast and convenient, and is often used in the detection of several immunoglobulins in renal biopsy tissue and the detection of pathogens. But its disadvantage is that only one corresponding substance can be detected, the sensitivity is poor, and the effect is sometimes unsatisfactory.

Indirect immunofluorescence method has strong sensitivity due to the increase of fluorescein antibodies bound to the antigen-antibody complex and the strong fluorescence brightness. Therefore, the indirect immunofluorescence method is more commonly used, and it is only necessary to prepare one species of fluorescent antibody, which can be applied to the labeling display of multiple primary antibodies.

2. Operation steps of immunofluorescence staining experiment
The protocol for immunofluorescence staining typically includes: fixation and permeabilization, blocking, primary antibody incubation, washing, secondary antibody incubation, rewashing, and assay readout. This article takes indirect immunofluorescence as an example to explain the principle of each step from fixation to mounting.

1) Sample preparation
For adherent cells: you can directly use multi-well plates, such as 6-well plates, 24-well plates, etc., to culture cells, and then perform subsequent operations such as fixation at a predetermined time. You can also use a clean coverslip, soak it in 70% ethanol, place it in a 6-well plate with sterile tweezers, and then wash off the residual ethanol with sterile saline, PBS or culture medium. At this time, cells can be seeded for culture, and after the cells have grown well on the coverslip, subsequent operations such as fixation can be performed.

For suspension cells: fix the cells in fixative first, then drop the cells on the slide, and the cells will stick to the slide after drying. Then follow-up operations can be performed. If the adhesion of the cells is poor, the slides can be treated with substances such as PDL to enhance the adhesion of the slides.

For cryosections: After the sections are placed on the slide, subsequent operations such as fixation can be performed directly.

For paraffin sections: Deparaffinize sections in xylene for 5 min, then deparaffinize with fresh xylene, and deparaffinize 3 times with shared xylene. Absolute ethanol for 5 min, twice. 90% ethanol for 5 minutes, twice, 70% ethanol for 5 minutes, once. Distilled water for 5 minutes, twice.

Antigen retrieval: According to different antigens and antibodies, the sections can be placed in the following antigen retrieval solution, 10mM sodium citrate, pH6.0, or 1mM EDTA, pH8.0, or 10mM Tris, pH10.0, heated at 95℃ 12 minutes, cooling slowly to room temperature in about 30 minutes.

2) Fixed
The goal is to preserve the original structure of the tissue and maintain cell morphology and cellular distribution of antigens. When tissue cells die, the cells break down, and enzymes released from their lysosomes and other organelles can hydrolyze the tissue, a process called autolysis. To avoid this, it is necessary to fix the tissue cells.

Cells or sections can be fixed with an appropriate fixative and, after fixation, washed twice with Immunostaining Wash (P0106) for 5 min each. Commonly used fixatives are formaldehyde, glutaraldehyde, methanol/acetone. The time and concentration required for different fixatives vary and need to be explored according to specific experiments.

3) Membrane rupture
The membrane is ruptured so that the antibody can have a chance to meet the antigen. Because the basic principle of immunostaining is to combine antigen and antibody, and then the labeled antibody fluoresces, so that we can qualitatively or quantitatively analyze the target protein. If the antigen to be detected is on the outer surface of the cell membrane or the extracellular matrix, then the permeabilization step can be omitted. Because the antibody can also have the opportunity to bind to the antigen without breaking the membrane. However, if the antigen to be detected is intracellular, the membrane needs to be broken and the cell exposed to a primary antibody against the protein of interest to ensure that the antibody can access the epitope. Commonly used membrane breaking agents are Triton X-100, NP-40, Brij-58, saponin, digitonin, methanol/acetone. Again, the choice of membrane-breaking agent should be made according to the specific experiment.

4) Blocking
Blocking is an important step to minimize nonspecific binding of the primary antibody. In the process of using a specific primary antibody to bind to the target protein, if there is non-specific binding, false positive results may occur, and strong background staining will also interfere with the presentation of the target staining and affect the judgment of the experimental results. In theory, any protein that does not bind the target antigen can be used for blocking. Serum is a common blocking agent because it contains antibodies that bind to nonspecific sites. Blocking with serum or protein blocking agents prevents nonspecific binding of antibodies to tissue or Fc receptors.

All steps starting from the blocking, must pay attention to the moisturizing of the sample to avoid drying of the sample, otherwise it is easy to produce a high background.

5) Primary antibody incubation
Pre-selected specific primary antibodies can bind to the protein of interest. In this step, you need to pay attention to what species the primary antibody is against. If the tissue cell source is mouse, the primary antibody should be some kind of animal anti-mouse, where a certain animal refers to the primary antibody host, for example, the primary antibody is goat anti-mouse, and the goat is the primary antibody host.

6) Secondary antibody incubation
After the primary antibody is incubated, the unbound primary antibody is washed away, and the binding of the primary antibody to the secondary antibody can be carried out. The secondary antibody should be a fluorescently labeled secondary antibody against the primary antibody host species. For example, the primary antibody is goat anti-mouse, and the secondary antibody should be some kind of animal anti-goat, such as rabbit anti-goat.

If double staining is performed, attention should be paid to the selection of the primary antibody and the secondary antibody host, so that there is no possibility of cross-binding. For example, if the tissue source is mouse, and there are two target proteins to be detected, the primary antibody host should be different, and the secondary antibody should have different fluorescent labels. The primary antibody against target protein A is goat anti-mouse, the primary antibody against target protein B can be rat anti-mouse (rat and goat are different primary antibody hosts), and the secondary antibody against A is a rabbit antibody with green fluorescence Goat secondary antibody, B secondary antibody is a rabbit anti-rat secondary antibody with red fluorescence, this combination is possible. Under the fluorescence microscope, target protein A was stained green, and target protein B was stained red. However, if the B primary antibody is also a goat anti-mouse, the A secondary antibody will also bind to the B primary antibody, which is messed up at this time, and it is green under the fluoroscope.

7) Coverslip
Mounting refers to the use of mounting medium to adhere coverslips to tissue sections or cell smears after immunostaining. Coverslips protect stained specimens from physical damage. The mounting medium in which the coverslips are performed also helps to improve the clarity and contrast of the image under the microscope.

8) Detection of proteins
For immunofluorescence staining, it is already possible to observe directly under a fluorescence microscope at this point.

​How to solve the strong background color of immunofluorescence staining? Explain the experimental operation and precautions in detail

3. Examples of three kinds of cell immunofluorescence staining experiments
Immunofluorescence of zo-1
1) Remove from the incubator when the cells grow to 95%-100% confluence on the coverslip.
2) Wash 3 times with pre-warmed 1×PBS for 10 minutes each time
3) Fix in 4% formaldehyde at room temperature for 20-30 minutes
4) Wash 3 times with 1×PBS for 10 minutes each time
5) Permeabilize with 0.2% Triton X-100 for 2-5 minutes
6) Wash 3 times with 1×PBS for 10 minutes each time
7) Block with 5% BSA at room temperature for 30 minutes
8) Add primary antibody (diluted with 1% BSA) and put it in a humid box, overnight at 4 degrees
9) Wash 3 times with 1×PBS, 10 minutes each time
10) Add secondary antibody (diluted with 1% BSA) for 30 minutes, turn off the light! !!
11) Wash 3 times with 1×PBS for 10 minutes each time
12) Mounting with 95% glycerol
Note: 4% formaldehyde, 0.2% Triton, 5% BSA were all diluted with 1×PBS”

Immunofluorescence of cell slides
1) Take out the cell slide and put it in a 35mm or 60mm used petri dish, and wash it three times with PBS.
Sometimes the cell slides made may be relatively small, so be careful when picking, pay attention to the reverse side, it is more convenient to wash in a dish, avoid back and forth clipping, and add PBS when washing, do not wash too much, do not wash cells down. When washing, I always add more PBS, shake it a little and pour it out, without waiting for 5 minutes or 10 minutes.
2) Fixed in 4% cold paraformaldehyde for 20 minutes and washed three times with PBS.
3) Permeabilize with 0.2% Triton X-100 for 10 minutes, and wash three times with PBS.
4) The serum of the same host as the secondary antibody was blocked for 30 minutes, and washed three times with PBS.
5) The primary antibody can be stored overnight in a humidified box at 4 degrees, or at 37 degrees for 2 hours. It feels that the former is effective. Wash it three times with PBS.
6) The secondary antibody is kept at room temperature for 2 hours (in the dark), or 37 degrees for 1 and a half hours, and washed three times with PBS.
7) It is best to stain the nucleus with DAPI, and then directly photograph the fluorescent film.
8) Wash off the PBS with distilled water, seal the film with glycerin, and seal around the film with nail polish. Because glycerin does not dry like resin, it will be a mess if you don’t use nail polish to seal it.

Cellular Immunofluorescence Simple Experiment
1) Rinse serum protein H7.2-7.4 in 37 degree PBS for 2 hours.
2) After fixing in -20 degree methanol for 20 minutes, let it dry naturally for 10 minutes
3) Wash with PBS: 3min*3
4) 1% Triton: 25min-30min. Make into 50ultriton+5ml pBS
5) PBS wash: 2*5min
6) Goat serum blocking: 37 degrees, 20 minutes
7) Primary antibody, overnight at 4 degrees, generally more than 18 hours or 37 degrees for 1-2 hours
8) Wash with PBS at 4 degrees, 3min*5 times
9) The secondary antibody is less than one hour at 37 degrees
10) Wash with PBS at 37 degrees, dry for 3*5min and seal (blocking solution PH8.5)

No matter what method is used, when rinsing with PBS buffer, it must be rinsed clean and the pH value must be measured. You can use PBS to wash several times, or you can extend the PBS washing time. If the background of the result is high, you can extend the number of rinsing. and time.

​How to solve the strong background color of immunofluorescence staining? Explain the experimental operation and precautions in detail
4. Precautions for immunofluorescence staining
1) Fluorescent reagents should be put away

Although many fluorophores are relatively photostable, failure to protect from light during storage and staining may lead to degradation of the fluorophore-antibody conjugate, resulting in false negative results. Therefore, fluorescent substances must be carefully stored at recommended temperatures and always protected from light to protect their spectral integrity.

2) Choose Fluorescence Carefully
A major advantage of immunofluorescence technology is that it provides the opportunity for multiplexing, and today flow cytometry can measure more than 20 discrete parameters per cell. When designing multiplex experiments, the unique properties of each fluorophore, such as absorption and emission maximum wavelengths, extinction coefficients, and Stokes shifts, should be considered.

3) Appropriate controls are essential
Analysis of any experimental data relies on relevant controls, and immunofluorescence staining is no exception. For each test, the following three controls need to be set:
(1) Positive control: positive serum + fluorescent marker
(2) Negative control: negative serum + fluorescent marker
(3) Fluorescent marker control: PBS+fluorescent marker

5. Troubleshooting guide for common problems in immunofluorescence experiments
1) Background staining is too strong
Too strong background staining means that in addition to the specific staining we want to see playing the leading role in the foreground, there are also a bunch of melon eaters (the same color as the specific staining that we want to see) in the back.

What caused these actors to steal the protagonist’s play? Possible reasons are as follows.

The tissue section is too thick: In this case, you can choose to make the tissue section thinner.

Poor blocking: The purpose of blocking is to reduce non-specific binding and reduce background staining. If the background is too strong, consider changing the blocking solution or increasing the blocking incubation time

The secondary antibody has non-specific binding: to verify whether this is the case, you can make a blank control with only the secondary antibody added during the staining process (that is to say, do not use the specific primary antibody for the primary antibody incubation process, such as using primary antibody diluent. Carry out the step of incubating the primary antibody). If the blank control is stained, it means that the secondary antibody has non-specific binding. At this time, it is recommended to replace a secondary antibody.

Autofluorescence: To know if the tissue has autofluorescence, look for fluorescence in non-stained areas. Some of the autofluorescence comes from the fixation step, and glutaraldehyde fixation can be avoided or washed with 0.1% sodium borohydride in PBS to remove free aldehyde groups. There is also some fluorescence from endogenous fluorescent molecules that can be photobleached with Sudan black/copper sulfate.

Antibody concentration too high: decrease the antibody concentration used or adjust the incubation time.

Insufficient washing: There are many steps in dyeing, which in turn include many washing steps. During the experiment, make sure the washing is in place and do not cut corners.

2) Weak or no staining
The possible reasons are as follows: the target protein you want to study does not exist in the tissue itself or the expression level is very small.

If it is suspected that the protein of interest does not exist, it can be verified by various methods, such as WB. Because the principles and operations of different protein-exploring experiments are different, the results may be different, and the verification of multiple experiments is more likely to avoid false negatives. If the protein of interest is expressed in small quantities, an additional step to amplify the fluorescent signal can be considered.

Problems with fluorescence microscopy.

If the parameters of the fluorescence microscope are set incorrectly, the fluorescence may not be seen. For example, the light source/filter device does not match the fluorescence you want to detect. Every time you take a photo, remember to check if the parameters are correct.

Exposure time is too short or light absorption is too low (gain value is too low): You can try to increase the Gain value and/or increase the exposure time to find the best value.

Exposure time is too long and fluorescence quenching occurs: avoid overexposing sections. Store sections in the dark immediately after use.

Over-fixation of cells/tissues: Because over-fixation can make the epitope masked, so that the antibody cannot bind to the antigen, so of course there is no fluorescence. So you can try to reduce the fixed time, you can also try to do antigen retrieval to reveal the antigenic epitope.

Tissue/Cell Drying: Make sure the sections are kept moist throughout the staining process.

If the cell is not permeabilized, the primary antibody cannot enter: For example, if the target protein you want to study is in the cell, but the antibody cannot enter the cell and stay with the target protein because there is no diamond, then of course you cannot see them. sparkle of love. So, we can find a way to make the cells open green channels and build a bridge of magpies. For example, if formaldehyde is used to fix tissue cells, cells can be permeabilized with 0.2% Triton X-100 (concentration may vary between experiments). For tissue cells fixed with methanol or acetone, Triton X-100 is not required, as the former can permeabilize cells.

Insufficient amount of primary antibody/incubation time too short: increase the concentration of antibody or increase the incubation time

The primary antibody is not suitable: Before purchasing the primary antibody, remember to read the product information, not all primary antibodies can be used for immunofluorescence staining experiments. Also, make sure the product has not expired.

Incompatible primary antibody and secondary antibody: For example, if your tissue source is mouse, then the primary antibody should be some kind of animal anti-mouse, such as goat anti-mouse, then the secondary antibody should be some kind of animal antibody goat, such as rabbit anti-goat. That is, the secondary antibody should be against the primary antibody host.

Sections are stored for too long: the sample should be observed and photographed as soon as possible after staining, otherwise the signal will weaken over time. Consider storing slices at 4⁰C away from light for as long as possible.

Antibody storage issues: Avoid repeated freezing and thawing of antibodies as they may cause antibody degradation. It is recommended that after purchasing the antibody, according to the needs of each experiment, the antibody is divided into several small portions and stored, so that one small tube can be used each time. In addition, remember to read the instruction manual before storing, and store it according to the instructions in the instruction manual. Such as the specific storage temperature, whether to avoid light and so on.