Misunderstanding of immunofluorescence analysis

Recently, many fans have successively asked questions about “immunofluorescence” in the background, basically asking questions about immunofluorescence analysis.

Here comes the problem.

“Should immunofluorescence be analyzed?”

Regarding this topic, I would like to discuss it carefully in a question-and-answer manner. In this way, people who read this article will not feel confused.

Question 1: What is semi-quantitative analysis?

Answer: For some indicators, we cannot detect the absolute content. We can only set a reference indicator to reflect the relative content of the target in multiples of the reference indicator. This “relative” is for reference. The analysis result is semi-quantitative and unitless, because it is essentially a ratio (target/reference).

Therefore, the content of the reference index is constant under any circumstance, otherwise it will be a sword. The meaning of this sentence can be extended a bit, that is, as long as there is a constant known quantity in the test object, it can be used as a reference for semi-quantitative analysis.

Question 2: Illustrate which indicators are typical semi-quantitative analysis?

Answer: ① In Western Blot, ELISA, and RT-PCR experiments, housekeeping proteins such as GAPDH and β-actin are often used as internal references to analyze the relative expression of the protein.

② Immunohistochemical staining (DAB method), immunocytochemical staining (DAB method). It also uses housekeeping proteins such as GAPDH and β-actin as a reference, and collects images in bright field, and then uses Image J or Image Pro Plus to semi-quantitatively analyze the average optical density of positive expression products.

Note the red text above. The DAB staining method is observed under a bright field, that is, under a light microscope, and does not involve the fluorescence excitation process. The DAB stained sections will not fade. That is to say, the observation of the slices after DAB staining will not be affected by time, place, and observer changes, and the intensity and range of its positive expression will remain unchanged after mounting. This is the fundamental reason why semi-quantitative analysis can be performed after DAB staining.

Question 3: Is protein immunofluorescence suitable for semi-quantitative analysis?

Answer: Not suitable. The key to immunofluorescence staining is the fluorescent secondary antibody, and then take pictures under the fluorescent field and keep the pictures. On the surface, this process is similar to DAB staining, but in fact there are quite a few variables, and it is these variables that make immunofluorescence staining unsuitable for semi-quantitative analysis.

Variable ①: The quality of the fluorescent secondary antibody determines the fluorescent staining effect. The same slice and cell slide, using domestic fluorescent secondary antibody and imported fluorescent secondary antibody, there is a very big difference in effect, and everyone who has used it knows. At this point, can we say that the protein expression level is different based on the fluorescence intensity and distribution range? Obviously not.

Variable ②: Fluorescence shooting conditions. The shooting environment, microscope brand and excitation fluorescence determine the effect of observation under the microscope. The same operation method and slices show different fluorescence intensities under different brands of microscopes. But in fact, the protein on the slice never changed, what changed was the shooting conditions.

Variable ③: fluorescence attenuation. The fluorescence decay is very obvious. The same set of slices, taken within 1 hour and taken 8 hours later, the fluorescence effect is obviously different. For another example, when analyzing 50 fluorescently stained slices, the fluorescence intensity has changed when the first slice is analyzed and the 50th slice is analyzed. Although there are commercial anti-fluorescence decay reagents, these reagents can only fluoresce dyeing and will not decay rapidly in natural environments. After fluorescent dyeing, under the direct action of excitation light, it will decay rapidly in about 10 seconds. Friends who have taken photos should have the impression that after the excitation light is irradiated on the tissue slice, a circular area with dim fluorescence will soon be formed, which is the performance after fluorescence attenuation.

Variable ④: γ value (gamma value). In some cases, beginners often make the mistake of taking fluorescence photos under different gamma values, or adjusting the gamma value γ to present different fluorescence intensities. In this way, even the fluorescence intensity on the same fluorescent dye photo will be adjusted to a completely different effect when γ value = 0.75 and γ value = 0.8. In the detection of trace proteins, this change will fundamentally subvert the conclusion. Therefore, all fluorescence photos should be analyzed under the same gamma value.

Variable ⑤: measurement index. If, I mean, if you really want to do semi-quantitative analysis of fluorescence staining, which index should you use for analysis? Fluorescence staining area? Obviously not. The only theoretically possible method is to convert the fluorescent dyed color image into a black and white grayscale image, and then use the grayscale value as the measurement index. However, this method still does not circumvent the aforementioned variables, and its analysis results are still unreliable.

With so many variables, do you dare to perform semi-quantitative analysis of immunofluorescence staining?

Question 4: Why can WB protein bands be semi-quantitatively analyzed, but protein immunofluorescence is not?

Answer: Theoretically, the operations involved in obtaining a protein band are the same. The subsequent luminescence and photographing of the protein are also performed at the same time. Does not involve the issue of time difference. That is, you are testing different holes on this strip under the same conditions and at the same time. Although there is also the problem of signal attenuation, at this time, the fluorescence attenuation degree of all the wells representing the protein is the same.

Fluorescence staining is different, it takes longer to acquire images, and you cannot take all the slices at the same time. This brings a very obvious time difference problem, which also magnifies the influence of fluorescence decay on the results.

Question 5: Why can TUNEL fluorescent staining be analyzed again?

Answer: TUNEL fluorescence staining analysis is the gold standard for detecting cell apoptosis. It is by connecting the luminescent substance to the 3’end of the broken DNA. It is not an absolute immune reaction process. The essence of TUNEL fluorescence staining analysis is a counting problem, that is, by counting the number of apoptotic cell nuclei, the apoptosis index on the tissue section or cell slide is analyzed. In this process, our count will not be strongly affected by fluorescence decay. As long as it is judged that the fluorescent signal does belong to the apoptotic nucleus, regardless of the size of the signal point, as long as there is a signal, we can count as an apoptotic nucleus.

But immunofluorescence is completely different. The expression level of the protein is directly reflected by the intensity and distribution range of the fluorescent signal. Once the signal attenuates, the fluorescence of the positives will attenuate and the distribution area will decrease. At this time we can no longer evaluate the protein expression level.

Question 6: Since immunofluorescence is not suitable for semi-quantitative analysis, what role does it have?

Answer: In essence, immunofluorescence is the most suitable indicator for positioning analysis. We can merge on the same slice with different fluorescent secondary antibodies under different excitation light to produce different staining effects. In this way, we can evaluate the distribution range and spatial relationship of the studied protein. This is very important for studying the ultrastructure of cells or protein receptors. Conventional DAB dyeing simply cannot meet the requirements.

Question 7: How to maximize the strength of evidence when studying proteins?

answer:

① PCR detects the gene expression level corresponding to the protein;

②Western Blot as the main protein semi-quantitative analysis method;

③Use immunohistochemical staining or immunocytochemical staining (DAB method) to further evaluate the location of protein expression (extracellular, membrane, cytoplasm, nucleus), and analyze protein expression level by semi-quantitative analysis to consolidate Western Blot semi-quantitative analysis results;

④Using immunofluorescence method to re-evaluate the spatial distribution of the target protein to consolidate the evaluation of the spatial distribution of the protein during immunochemical staining, and at the same time enrich the color of the entire study;

⑤ If necessary, the above four methods can be further used to study the receptors of the target protein, and to clarify the relationship between upstream and downstream and the same level of regulation.