IHC principle, operation and precautions
Immunohistochemistry (IHC) is a technique that uses the principle of specific binding between antigens and antibodies to locate, characterize and quantify antigens (peptides and proteins) within tissue cells by chemically reacting the marker antibody's color developer (fluorescein, enzyme, metal ion, isotope) to produce a color reaction.
The experimental procedures and methods of IHC are generally not difficult, but it is not easy to achieve beautiful staining results. As the saying goes, “details determine success or failure,” and this is also a key point that is particularly important in IHC experiments. So what are the main “details”? I will explain them to you below. (The following is an introduction using paraffin tissue sections as an example)
1. Specimen fixation
The purpose of fixation is not only to solidify the proteins in the cells, reduce or terminate the reaction of endogenous or exogenous intracellular decomposing enzymes, and prevent autolysis of tissue cells, but also to preserve the antigenicity in the tissues or cells, so that the antigens do not become inactive and disperse. Different antigens have different degrees of tolerance to fixative, so it is necessary to choose the appropriate fixative.
Common fixatives include neutral formalin and 4% paraformaldehyde-phosphate buffer. Some fixatives have better effects on specific tissues. For example, picric acid has a softening effect on scalp and nail fixation, Helly fixative has a better effect on islets of Langerhans and pituitary glands, and PLP fixative has better antigen preservation for sugary tissues.
2. Dehydration, paraffin embedding and sectioning
Dehydration: Use a gradient of ethanol (from low to high) to fully dehydrate the tissue. For some fragile tissues (such as liver and spleen), the time in the high concentration of alcohol should be reduced, and the transparency time should also be controlled and shortened. When immersing the tissue in wax, paraffin with a melting point of 56°C to 58°C is generally used. The temperature of the waxing should preferably not exceed 60°C to prevent loss of antigens. The embedding should be done quickly at a chosen angle to ensure a complete section when cutting. When cutting, the speed should be adjusted according to the situation, and the blade should be checked in time to prevent scratches on the wax strip.
3. Dewaxing and hydration
Restores the tissue to its normal state after fixation, exposing the antigen to facilitate binding with the primary antibody. If the dewaxing and hydration are not complete, focal reactions and incomplete immersion may occur, resulting in nonspecific background staining.
4. Antigen repair
During formalin or paraformaldehyde fixation, cross-linking between proteins and the blocking effect of aldehyde groups occur, thereby masking antigenic determinants. Antigen repair makes intracellular antigenic determinants re-exposed, thereby improving the antigen detection rate. Common repair methods are generally divided into three types from strong to weak: high-pressure heating repair, microwave repair, and trypsin repair. Among them, high-pressure heating repair is a simple and easy-to-operate method with better results. When using high-pressure heating for repair, it is important to grasp the repair temperature and time. The higher the temperature, the shorter the repair time, and the temperature is negatively correlated with the repair time. After the repair is complete, pay attention to cooling at room temperature to allow the protein to renature naturally. Secondly, try to use an excess amount of antigen repair solution to prevent the high-temperature liquid from evaporating and drying out, which can cause irreversible damage to the section.
5. Inactivation of endogenous peroxidase and biotin
In the traditional ABC method and SP method, immunohistochemical reactions are easily interfered with by endogenous peroxidase and biotin, which must be inactivated and blocked with hydrogen peroxide and ovalbumin. Endogenous peroxidase is usually inactivated with 3% hydrogen peroxide for about 10 minutes. Methanol-formulated hydrogen peroxide is more suitable for protecting antigens and fixing tissues.
6. Serum blocking
To prevent non-specific binding of the primary antibody to the tissue, which can cause false positives, these sites are usually blocked with BSA, goat serum, etc. The blocking serum is usually from the same animal source as the secondary antibody and is blocked at room temperature for 10-30 min.
7. Primary and secondary antibody concentration and incubation time
Different primary antibody concentrations, incubation times and temperatures often have a significant effect on the staining results. At 4 °C, the reaction is slow and the background is lighter; at 37 °C, the reaction is faster and shorter; room temperature is between the two, and incubating at room temperature is conducive to the convenience of the experimental process and is also suitable for the detection of a variety of samples. The secondary antibody is usually incubated at room temperature for 30 minutes.
8. Section cleaning (soaking, rinsing and rinsing)
In order to prevent non-specific staining caused by residual primary and secondary antibodies, it is particularly important to wash appropriately. It is recommended to use a washing bottle to rinse for 30 s X 3 times, and the primary antibody can be washed separately with TBST after incubation. During washing, pay attention to separate rinsing to prevent contamination caused by cross-reactions, and rinse gently to prevent detachment of sections. The pH and ionic strength of TBS are recommended to be 7.6-8.0, and the concentration is 0.01 M. 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. DAB staining
The intensity of the background and the intensity of the specific staining can both be determined by the incubation conditions of DAB. The DAB staining time is not fixed, and it is mainly controlled under the microscope. When the specific staining is strong and the background coloring is light, the sample can be rinsed. If the DAB staining time is very short (such as a few seconds or tens of seconds), a very dark brown color appears, which is likely to indicate that the concentration of the primary antibody is too high and needs to be adjusted downwards; if a dark background appears after a very short time, it is possible that non-specific protein blocking was not complete, and the blocking time needs to be extended; if positive staining only appears after a long DAB staining time (e.g., more than ten minutes), it may be that the concentration of the primary antibody is too low or the blocking time was too long. For weak DAB colors, enhancement methods can sometimes be used. For example, adding metal ions: copper sulfate, hexamethylenetetramine silver, cobalt chloride, ammonium nickel sulfate, nickel chloride, and imidazole.
10. Counterstaining
Immunofluorescence staining must be counterstained to bring out the morphological structure of the tissue. Mayer's hematoxylin counterstaining is currently commonly used, generally for about 2 minutes. If DAB stains the nuclear protein, the counterstaining time can be appropriately shortened, and then ammonia or TBS at pH 8.0 is used to remove the blue color.
11. Cover slips
For long-term preservation, neutral gum is usually used to seal the slides. During the sealing process, be sure to lean the coverslip at an angle to prevent bubbles from affecting observation. If the gum is too viscous, you can add an appropriate amount of xylene to dilute it, so that the gum can spread quickly in the seal. After dehydration, it is recommended to seal wetly, that is, seal the slides while the xylene is still on the tissue, and be sure to operate in a ventilated cupboard. This helps to exclude bubbles cleanly and does not affect subsequent microscopic inspection.
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