Immunofluorescence (IF) how to make pictures more colorful

Junior: Brother, brother, come out soon

Senior: What should I do to my sister and sister, little sister

Junior: The fluorescence of IF I made with NeuN antibody is not clear, is it the reason for the secondary antibody?


Senior: NeuN is a mature nuclear antigen, which focuses on nuclear localization. There are many reasons for the blurring of fluorescence. Judging from the results, the causes of antibodies can be eliminated.

Junior: We generally think that it is the cause of antibodies if the experiment is unsuccessful. Why is this not the cause of antibodies this time?

Senior: That’s for sure! Let me tell you slowly.

Each picture of immunofluorescence is a work of art. If you are a senior player of experimental immunohistochemistry, you can take a picture of it like this:

protein extraction

It is from

protein extraction

It is from

protein extraction

Various tissue shapes, cell structures, and protein shapes are scattered and organically combined to form a colorful and exquisite fluorescent world, uncovering the mysteries of scientific research one after another. It’s really beautiful (du).

So, how to operate in order to obtain a series of perfect immunofluorescence results like a senior player?

Today, I will take you to take a closer look at the immunofluorescence experiment~

Experimental principle:

Immunofluorescence (immunofluorescence technic): Coons equal to the first use of fluorescein for labeling in 1941 and achieved success. This technique of labeling antibodies with fluorescent substances for antigen localization is called fluorescent antibody technology (fluorescent antibody technique). The method of tracing or checking the corresponding antigen with fluorescent antibodies is called the fluorescent antibody method; the method of tracing or checking the corresponding antibodies with known fluorescent antigen labels is called the fluorescent antigen method. These two methods are collectively referred to as immunofluorescence technology, because fluorescent pigments can not only bind to antibody globulin for detecting or localizing various antigens, but also bind to other proteins for detecting or localizing antibodies, but fluorescent antigens are used in actual work. Technology is rarely used, so people are accustomed to call it fluorescent antibody technology, or immunofluorescence technology. The fluorescent antibody method is more commonly used. Immunofluorescence technology to display and examine the antigen or hapten material in cells or tissues is called immunofluorescence cell (or tissue) chemical technology.


One. Sample processing

 Cell slide

  1. In the cell culture well plate, the slides that have climbed the cells are rinsed with PBS 3 times, 5 min each time.


  1. Fix in 4% paraformaldehyde or other fixing solution at room temperature for 15 minutes, wash three times with PBST for 5 minutes each time (fixation method is not unique, depending on the total cell type).

Frozen section

  1. Frozen tissues of fixed tissues were equilibrated and rewarmed at room temperature for 30 minutes;
  2. Wash three times with PBST, 5min each time;

Ø Paraffin section

  1. Dewaxing Hydration
  2. Soak in xylene Ⅰ cylinder for 15min;
  3. Immersed in xylene II tank for 15min;
  4. Soak in the No. 1 cylinder of absolute ethanol for 5min;
  5. Soak in absolute ethanol No. 2 cylinder for 5min;
  6. Soak in 95% ethanol for 5min;
  7. Soak in 80% ethanol for 5min;
  8. Soak in 60% ethanol for 5min;
  9. Wash with deionized water 3 times, 5min each time.

Here, cylinders I and II refer to different containers, but the contents are the same.

  1. Antigen retrieval

Take an appropriate amount of sodium citrate repair solution (pH 6.0) or Tris-EDTA repair solution (pH 9.0) in a glass beaker (the repair solution can not pass the slice holder), after the repair solution is boiled, put the slices in it, Close the lid and continue to boil the repair solution for 15min. Cool naturally in a fume kitchen.

two. Antibody recognition and fluorescence photography

  1. Wash the slides 3 times with PBST, 3 minutes each time, absorb the PBST with absorbent paper, add normal goat serum onto the slides, and seal at room temperature for 30 minutes;
  2. Absorb the blocking solution with absorbent paper, do not wash, add enough diluted primary antibody dropwise to each slide and put it in the wet box, incubate at room temperature for 2-3h (or incubate overnight at four degrees); secondary antibody incubate at room temperature Overnight at 1h or 4°C. Note: Starting with the addition of fluorescent primary antibodies, all subsequent steps should be carried out in a dark place.
  3. Counterstaining nucleus: add DAPI and incubate in the dark for 5min to stain the specimen, PBST 5min × 4 times to wash away excess DAPI; 4. Absorb excess liquid on the slide with absorbent paper and quench with anti-fluorescence The mounting solution of the agent is mounted, and then observed and collected under a fluorescence microscope.

Troubleshooting FAQ

  1. What should I do with irregularly shaped bright spots captured by immunofluorescence microscopy?

Irregular and more bright spots are generally due to impurities or dust on the surface of the slide material, which can be avoided by adjusting the focal plane to the tissue.

  1. What is the method of channel switching during the photo?

First find the target position under white light, and adjust to the clearest focal length, and then switch to the fluorescent channel to shoot.

  1. How to avoid dye fading?

In order to reduce the degree of fading of some samples, it is recommended to use neutral density filters in the light path before the light reaches the excitation filter, thereby reducing the intensity of the excitation light. In other cases, the fading effect can be reduced by changing the pH of the mounting medium or by using anti-bleaching agents (a few more important agents are listed in Table 2). For digital imaging, microphotography or simple visual observation, changing the field of view quickly can also avoid the fading effect.

  1. What is the reason why the organization is clear in the process of taking pictures?

In the process of making tissue sections, there are some areas that are not firmly attached to the slides, and there is a tendency to take off the slide, resulting in the sample not on a focal plane, and a blurred image under the microscope.

  1. No staining of tissue cells

One case is experimental operation problems, including inaccurate experimental operation methods, stale experimental materials, antibody species and reactivity selection, and antibody quality problems. These can be eliminated by setting up positive and negative control experiments. The second situation is the protein expression problem. Like the “WB experiment without bands” mentioned in the previous issue, the IF experiment preparation stage also needs to consult a lot of data to initially confirm the spatiotemporal expression profile of the target protein (including expression amount, expression site, and stimulation conditions Wait).

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