How to do immunohistochemistry as you like?
Immunohistochemistry has been in existence for more than 80 years, and the theory of immunohistochemistry was discussed from 1930. Until 1941, antibodies against fluorochrome were successfully observed in tissues. The existence of this, and then continue to improve the method, it has formed the immunohistochemistry technology used in our experiments, let's talk about why immunohistochemistry is still important in the laboratory for more than 80 years. One of the experimental projects.
The organs of the human body are composed of the smallest unit of cells. In general experiments, whether it is to study proteins or genes from cells, we often want to know that after the cells of the smallest unit are amplified into tissues, the proteins or genes we study will be What kind of way is presented in the organization? At this time, it is not difficult to understand the importance of these two techniques, whether it is research protein (immunohistochemistry) or research gene (chromogenic in situ hybridization, CISH).
We first discuss the experiment, and the headaches that the general collaborators will encounter will be the background. The background is generated in all directions, from pre-tissue treatment, antigen retrieval, and immune blockade. The final coloring, all of which are likely to cause background, so it is very important to carefully select a good set of immunohistochemistry for the whole experiment.
Let's discuss how the background is generated one by one. From the pre-organizational processing, we first get the organization, we will go through a step (perfusion), why should we perfuse it? We must first know that the most common method used by our immunohistochemistry is HRP coloration. Then we pull the lens back to our organization. We need to know that the most abundant HRP in the tissue is our blood cell. From this we can probably know that if we don't clean the blood cells, it will easily cause subsequent coloring. , we will find that the blood cells are not cleaned up, we will have the so-called background, and the other is to remove the blood cells in the tissue, which is the peroxidase remover that we will use in immunohistochemistry. Both are effective in removing HRP from our organization.
The other step is also a step that will be used in group experiments but often overlooked: antigen repair, why do antigen repair? The reason is that when we get a fresh sample, the step of formaldehyde fixation is usually carried out. In the process of formaldehyde fixation, an aldehyde bond is formed. The aldehyde bond is a determinant group that blocks the antigen. This compound blocks the antigen, but the antigen is blocked. After that, when we perform antibody binding, it will cause false negatives because the antibody cannot recognize the antigen, which is why the antigen is repaired. There are many ways to repair the antigen. The most commonly used ones are the following three. Citric acid buffer (Citrate), EDTA buffer, enzyme (Enzyme), usually we use citrate buffer, mainly because it can carry out a wide range of antigen-recovering antigens, but there will still be The location of some antigens is not handled by citrate buffer, so it is also important to choose the correct antigen retrieval solution during the experiment.
Let's talk about the immune blockers that will be used in the experiment. There are many kinds of formulas for immune blockers, but in fact, each formula may only be removed for a specific background. In this case, a complex immunoblocker is selected. It will be more helpful for the experiment. In fact, we will find that most of the group experiments are based on the background of the mouse or rabbit. In fact, this question is not difficult to answer. We flipped through the antibodies in our refrigerator. Most of the species came from mice or rabbits. For this reason, there was a background in animal experiments, so the immune blockers Choosing to become a whole experiment is a big key.
Finally, we discussed the coloration, but this is related to the experimental rule. The DAB or AEC we use is colored. Their principle is to develop color by precipitation. This also means that the experiment we paint is more The longer the color will be, the more important the task of coloring is to complete the entire immunohistochemistry.
Combining the items discussed above, it is not difficult to organize. In fact, the devil is hidden in the details. We only need to deal with each part of each link that may produce background. The immunohistochemistry is a very simple experiment. .
Let's talk about a problem we will encounter in the design of immunohistochemical staining experiments. Generally, there is absolutely no protein to be studied in our design experiments. We will definitely look at a variety of proteins to let us study the theme of this story. Complete, but generally we can only dye a visible color, so if we want to observe the simultaneous inhibition of two proteins on the tissue or mutual growth, we will definitely use a dyeing method ( Double stained Double stain), I believe that everyone who is double-dyed in the room must be familiar, but you are often familiar with fluorescent double dyeing. At this time, there will be doubts. Why can you only do fluorescence? Mainly fluorescent double staining, we can use the different separation of the excitation light to shoot, and finally the synthesis of the picture, but this also leads to other problems. The position of the first protein, we first use immunohistochemistry is not In order to see where the protein we studied is represented? But if you use fluorescent staining, you will understand that only the position of our protein will glow, while the other parts are all black, so it is difficult to discuss whether it is correct in the organization's position, another problem photo The synthesis, we will discuss with double dyeing, we will first take the DAPI, which is the nuclear part, and then we will study the A protein and B protein. After the whole filming, the software will be synthesized. The first time will take a long time. The second point is that in the case of synthesis, if the background is not processed very cleanly and thus produces some non-specific combination of performance, then using three pictures to synthesize one at the same time, the result picture may become very bad for data. judgment.
In fact, everyone will have a question in their hearts, why not do visible light? In fact, according to the previous technology, it is really not good to carry out double staining of visible light, mainly because of the immunohistochemistry of visible light. At the same time, if the two antibodies are not processed, there will be a dirty background. I believe that all the colleagues who want to do it must be I have encountered the above problems, but in recent years, with the advancement of technology and methods, we have broken through the problems that will cause background, so as to face the problems caused by the above fluorescence, for example, we want to see two in the same field of vision. There is no background in the performance of the protein. It will be solved by this new technology. In the past, there was no way to do visible light before it was double-dyed. However, this problem has been solved. I believe that the immune group of visible light Double dyeing, this new technology can take everyone's research to the next level.
Finally, I would like to introduce the technique that is very popular in recent research (chromogenic in situ hybridization, CISH). This technology is not new. It was developed in the late 1980s, but it was studied at that time. The focus is on the DNA, but because the technical threshold is high and the success rate is not so stable, this technology is often seen in the book, but the actual application is actually not wide, so why is it said that it is the most popular recently? Get up? First, with the rapid development of the biotechnology industry, technology and academics are constantly evolving, and problems encountered in the early days can be easily solved. Secondly, microRNA has become a mainstream research direction. In the early days, we continued to research. With the continuous updating of academic knowledge, we continue to search for proteins from downstream products, and finally find the source of microRNAs. We first used real-time PCR to measure. But the measured results are a long series of data analysis, people's curiosity is unlimited, as the research time is longer, people's curiosity is higher, so return to the problem we mentioned at the very beginning, if there is a way The organization sees the performance of microRNAs. I don’t know what it will be. It’s the idea that the originally long-lived in situ hybridization method has instantly become a new star in the academic and research world. This method satisfies all our curiosity. With the improvement of technology, the in situ hybridization experimental procedure becomes easier, and the research direction is from the earliest DNA, RNA to
Organizational research is a research field that cannot be erased and replaced in academic research. We continue to research and develop new research techniques related to pathology, and we have always been at the forefront of research technology. In the future, we will be the most popular in the future. Two techniques of in situ hybridization and immunohistochemical double staining are performed simultaneously on the same tissue, which means that one organization can simultaneously see the performance of the gene and simultaneously observe the performance of the protein. I believe this emerging technology Once again, the technology of pathology will be pushed to a higher peak.
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