The immunofluorescence method was the first established immunohistochemical technique. It uses the principle of specific binding of antigen and antibody to first label known antibodies with a light element as a probe to examine the corresponding antigens in cells or tissues under a fluorescent microscope. When the fluorescein in the antigen-antibody complex is irradiated by excitation light, it emits fluorescence at a certain wavelength, thus allowing the localization of an antigen in the tissue to be determined and then quantitative analysis can be performed.

Because of its high specificity, sensitivity, rapidity and simplicity, immunofluorescence technique is widely used in clinical pathology diagnosis and testing.

1. The main steps in the immunofluorescence method:

1) Frozen section preparation: It is recommended to use fresh tissue otherwise the internal structure of tissue cells is easily disrupted to make the antigen diffuse. Choose a clean and sharp blade, the tissue must be frozen moderately, etc. to prevent lobing and deslicing.

2) Tissue section fixation: Immediately after cutting and air-drying, use ice acetone and other fixing solution to fix 5-10min, especially to preserve the white film for a long time, must be fixed and properly preserved in time.

3) Serum closure: In order to prevent the binding of endogenous non-specific protein antigen need to be closed with serum (and secondary antibody source) before the incubation of the first antibody to weaken the backstorm coloring, the time of serum closure is adjustable generally 10-30min.

4) The most important conditions for incubation of the primary antibody in immunohistochemical reactions include incubation time and antibody concentration. One antibody incubation check temperature there are several 4 degrees, room temperature 37 degrees which 4 degrees better effect incubation time which is related to the temperature, antibody concentration generally 37 degrees 1-2h4 overnight (from the refrigerator after taking out 37 degrees rewarming 45min). Specific conditions have to be figured out.

5) secondary antibody incubation conditions generally room temperature or 37 degrees 30min-1h specific time needs to be figured out and the concentration generally have working solution if it is concentrated solution also have to figure out the concentration must be protected from light reaction, but in immunofluorescence we generally first set the concentration of the three antibodies and incubation time first and then to figure out the concentration of the primary antibody and Bonjour time. Finally fluorescein-labeled secondary antibodies may have a large star of free fluorescein residue with the extension of storage time need to pay attention to the preparation of the small package and and carry out appropriate business heart.

6) The purpose of restaining is to form a cellular corridor so as to better localize the target protein, and generally DAPI restaining is commonly used.

7) Sealing: For long-term storage, we generally use buffered glycerol and other sealing solutions, in addition to special anti-fluorescence extraction sealing solution. The method to avoid bubbles is to put a drop of sealing solution directly on the slide and then hold one corner of the coverslip with one hand while holding the opposite corner with the other hand, and lower the corner near the proximal end of the sealing solution until it touches the liquid.

8) section cleaning: in order to prevent the primary antibody, secondary antibody and other reagents residual and cause non-specific staining so appropriate to strengthen the cleaning (extend the time and increase the number of times) is particularly important generally in the incubation of an antibody before the cleaning is 3min * 3 times and an antibody incubation after the cleaning are 5 times * 5min.

Note:
(1) Wash separately to prevent cross-reactivity and contamination.
(2) Rinse gently to prevent dislodgement of sections. I prefer to use the immersion rinse method;
(3) The rinsing time should be sufficient to thoroughly wash away the bound material.
(4) PBS PH and ionic strength of the use and requirements (recommended PH at 7.4-7.6, concentration is 0.01M; neutral and weakly alkaline conditions are conducive to the formation of immune complexes, while acidic conditions are conducive to decomposition; low ionic strength is conducive to the formation of immune complexes, while high ionic strength is conducive to decomposition)

9) Take pictures immediately if you can, but if you cannot take pictures in time, seal the film and seal it with nail polish, and keep it away from light and humidity. The use of fluorescence microscope attention in strict accordance with the fluorescence microscope factory manual requirements for operation do not arbitrarily change the procedure should be examined in a dark room to prevent ultraviolet light on the eye ingestion in the transfer of light source should wear protective glasses inspection time each time to 1 ~ 2h is appropriate more than 90min. ultra-high pressure recording lamp luminous intensity gradually decline fluorescence weakening specimens by ultraviolet radiation 3 ~ 5min after the fluorescence is also significantly weakened or faded excitation light for a long time. Or fading excitation light for a long time, will occur in the fluorescence of the decay and quenching phenomenon: so at most not more than 2 ~ 3h fluorescence microscope light source life limited specimens should be concentrated inspection, in order to save time and protect the light source.

When the day is hot, should be added to the fan cooling cooling new light bulbs should be used from the beginning of the record time. After the light is extinguished to re-enable the light to be fully cooled before ignition. Avoid lighting the light source several times. Turn off the mercury lamp at least 15-30 minutes after turning on the specimen stained immediately after observation because the fluorescence will gradually diminish over time. If the specimen is placed in a polyethylene plastic bag at 4 ℃ to delay the time of fluorescence weakening, to prevent the evaporation of the sealer, the use of slides and other carriers must be uniform thickness without obvious autofluorescence if you use oil mirrors must also ensure that the mirror oil for non-fluorescent mirror oil power better installed voltage regulator, otherwise the voltage instability will not only reduce the life of the mercury lamp will also affect the effect of microscopy.

2. What are the reasons for deeper background staining?

1) the antibody concentration is too high. Antibody concentration is too high is one of the common reasons. The solution is to test the working concentration of new antibodies before each use, so that each antibody individualized to find the ideal working concentration for their own laboratory even if it is a ready-to-use antibody should also do so, not just simply stain according to the instructions.

2) The antibody incubation time is too long or the temperature is high. The solution is to strictly implement the operating procedures and better carry a time table or clock to remind you to avoid prolonging the time due to forgetfulness. Nowadays, the popular two-step method (Polymer) is very sensitive and requires that the incubation time of the primary antibody is not the traditional 1 hour but 30 minutes, so it should be adjusted according to the staining results.

3) DAB deterioration and color development time is too long. DAB is better to be used now if the residue should be filtered and then used. DAB should not be stored for too long because in the absence of enzymes, hydrogen peroxide will react with DAB by releasing oxygen atoms and reduce the effectiveness of DAB. However, this may not seem practical when there are too many stained pieces or when using a staining machine, but at least some new or rarely used antibodies should be monitored to avoid prolonged staining times.

4) Tissue dryness is caused by failure to replenish the liquid in time after the repair solution has overflowed, too many stained sections, too slow movements, forgetting to drip the liquid, or loss of drops. The solution is to operate carefully using DAKO pen or PAPPen to draw circles around the tissue can effectively avoid the loss of liquid can also improve the operating speed.

5) slices in the buffer or repair solution soaking time is too long (more than 24 hours). The reason is not clear, but the phenomenon exists. Some laboratories like to dewax the sections before to repair the next day plus antibodies for immunohistochemical staining if the container with sections and repair solution is placed in the 40C refrigerator overnight has no significant effect on the results if placed at room temperature especially in the hot summer will appear background coloring Therefore, it should not be stored for too long.

6) the primary antibody deterioration, poor quality Dock drop antibodies. Pay attention to the expiration date of antibodies expired antibodies either not colored or background coloring, with newly purchased antibodies better set up a positive control and use used antibodies for comparison.

3. What are the reasons for not being colored?

1) paraformaldehyde fixation time is too long or not enough to adjust the fixation time

2) triton permeabilization triton permeabilization degree is not enough or triton permeabilization excessive

3) BSA incubation

4) primary antibody (you are sure that you add the amount of antibody is enough) is not enough to use or dilution ratio is too large to readjust and then re-stain

5) secondary antibody (you are sure that you add the antibody is enough) and the same as the first antibody to redilute or increase the amount used to re-stain

6) look at the fluorescence fluorescence intensity may not be sufficient to re-label the marker for analysis

4. What are the reasons for incorrect positioning?

1) the nucleus interferes with the cytoplasmic staining in front of the location of the nucleus causing interference can reduce the concentration of antibody incubation time.

2) the cell or tissue state is not correct cell or tissue state is different resulting in your target egg day cell positioning is different resulting in the final positioning is not correct if there has been positioning is not correct problem suggest to re-culture cells to adjust the state or re-take the material, staining again.

3) co-localization problem analysis assuming that you want to prove that a cell has undergone a certain change, in other words, there is both a protein expression and another protein expression two proteins belong to the co-localization of the situation can be detected by immunodouble labeling: if the two proteins do not belong to the co-localization. Suppose one is expressed in the membrane and one is expressed in the cytoplasm, this situation should not be co-localized and belongs to the co-expression, then the experiment should be redesigned to verify your conclusion.

4) nuclear localization is not the antigen expressed in the nucleus localization with immunofluorescence or immunoenzymatic labeling can be. If the positioning is not correct, it is recommended that the closure and perforation combined into one step, that is, the addition of 0.5% TRITON-100 in the closure solution 37 degrees closed for 2 hours plus an antibody after the best 4 degrees incubated overnight (16 hours).