1, the specimen is fixed
The purpose of fixation is to prevent the specimen from falling off the slide; 2 to remove the lipid that blocks the antigen-antibody binding, so that the antigen-antibody conjugate is easy to obtain good staining results; 3 the fixed specimen is easy to store.
2, dehydration, paraffin embedding and production
The dehydration is fully dehydrated with gradient ethanol (low to high), the tissue is completely immersed in wax, and the blade is clean and sharp when sliced. Otherwise, it is easy to split and peel off.
3. Dewaxing and hydration
This is because a reagent such as a later antibody can sufficiently bind to an antigen or the like in a tissue. Dewaxing can be 60 degrees for 20 minutes, then immediately xylene 1-3 for 10 minutes, but the slice made on the day can be 60 degrees 3-4 h. Gradient ethanol for hydration (high to low). If dewaxing and hydration are incomplete, focal reactions and impregnation may occur, resulting in non-specific background staining.
4, antigen repair
Because part of the antigen in the tissue is fixed in formaldehyde or paraformaldehyde, the cross-linking between proteins and the blocking of the aldehyde group occur, thereby losing the antigenicity; by antigen repair, the intracellular epitope is re-exposed and the antigen detection is improved. rate. Commonly used repair methods are generally divided into three types, from high to weak, high pressure repair, microwave repair, and pancreatic enzyme repair. The repair fluid is also divided into several types (specifically, relevant materials can be consulted, a large number: neutral, high pH, ​​etc.). Our laboratory generally uses microwave to repair medium fire 6min*4 times, and the effect is good. Pay attention to the natural cooling after microwave repair for about 30 minutes (as long as you think the temperature of the repair solution reaches room temperature).
5, the cell is transparent
The purpose is to enable the antibody to fully enter the cell for binding reaction. Generally, a permeate such as Triton X-100 or proteinase k is used. For example, Triton X-100 can dissolve lipids on cell membranes, nuclear membranes, and organelle membranes, allowing antibodies and macromolecular structures to enter the cytoplasm and nucleus. Therefore, it is recommended for use in cellular immunohistochemistry, so that antibodies can Smooth entry into the cell and binding to the corresponding antigen. Triton X-100 is typically used as a cell permeable agent in immunohistochemistry (>10 um thick sections) and immunocytochemistry to perforate the membrane.
6. Inactivation of endogenous peroxidase and biotin
In the traditional ABC method and SP method, the results of immunohistochemistry are easily interfered with by endogenous peroxidase and biotin, and must be inactivated with hydrogen peroxide and avidin. Inactivation of endogenous POD generally 3% hydrogen peroxide inactivation time is short, can be about 10min, while 0.3% hydrogen peroxide can extend the sealing time appropriately, generally 10~30 min; configure hydrogen peroxide to double steam with methanol Water or PBS may be good for protecting antigen and fixing tissue. If the incubation time of hydrogen peroxide is too long, it may cause stripping; now it is ready to be used, and it is stored at 4 degrees in the dark. However, there is now a "second-generation ready-to-use immunohistochemistry kit" to avoid interference from endogenous biotin, which is recommended.
7, serum closure
The remaining sites on the tissue section can be non-specifically bound to the primary antibody, resulting in a false positive of the subsequent results; the blocked serum is generally the same source as the secondary antibody, and the animal's own antibody in the serum can be cross-reactive with the tissue in advance. The site is combined; calf serum, BSA, sheep serum, etc. can also be used, but not consistent with the primary antibody source. Generally room temperature or 37 degrees 10-30 min.
8, primary and secondary antibody concentration and incubation time
Primary antibody incubation conditions are most important in immunohistochemistry, including incubation time and antibody concentration. The primary antibody incubation temperature is several: 4 degrees, room temperature, 37 degrees, of which 4 degrees is the best; incubation time: this is related to temperature, antibody concentration, generally 37 degrees 1-2 h, while 4 degrees overnight and taken from the refrigerator After 37 degrees, the temperature was rewarmed for 45 minutes. Specific conditions have to be explored. Secondary antibody incubation conditions: the secondary antibody is generally room temperature or 37 degrees 30 min-1 h, the specific time needs to be explored, and the concentration generally has working fluid, if it is concentrated solution, it is necessary to explore the concentration. However, in immunohistochemistry, we usually first set the concentration of the secondary antibody and the incubation time, and then explore the concentration of the primary antibody and the incubation time.
9, antibody dilution
In fact, many laboratory antibody dilutions can be used in general PBS, but in addition to the PBS component, special antibody dilutions are also added with sodium azide preservative, BSA stabilizer and other components, and multiple recycling of antibodies. better. For this reason, I have been using domestic proprietary antibody dilutions, and the results of the test have a negative result when replacing the new antibody dilution for a period of time (prompting that the primary antibody may not bind), and finally from the antibody concentration and incubation time, closure time After the reasons were excluded, it was found that the pH of the new antibody dilution was too acidic, and the antigen-antibody reaction was poor, and a false negative result eventually appeared.
10, slice cleaning (dipping, rinsing and rinsing)
In order to prevent non-specific staining caused by residual reagents such as primary antibody and secondary antibody, it is especially important to strengthen the cleaning (extended time and increase the number of times). I usually wash the primary antibody before incubation for 3 min*3 times, while the primary antibody Washing after incubation was 5 times*5 min. Note (1) Wash separately to prevent contamination from cross-reaction. (2) Gentle rinse to prevent the peeling of the slices. I like to use the dipping method; (3) the washing time is enough to thoroughly wash away the combined substances. (4) Use and requirements of pH and ionic strength of PBS. I have a painful lesson in this regard. At the time, the antibody dilution I bought was sour, and the background was yellow (no specific staining). It is recommended that the pH be 0.01 M at 7.4-7.6. (Neutral and weak conditions (PH7-8) are beneficial for the formation of immune complexes, while acidic conditions favor decomposition; low ionic strength favors the formation of immune complexes, while high ionic strength facilitates decomposition)
11, DAB color development
The depth of the background and the depth of the specific staining can be determined by the DAB incubation conditions. The DAB color development time is not fixed. The color development time is controlled by the microscope. When the specific staining is strong and the background color is light, the color can be washed. The DAB color development time is very short (such as a few seconds or tens of seconds). There is a very dark brown color, which may indicate that your antibody concentration is too high or the antibody incubation time is too long, you need to down-regulate the antibody concentration or shorten your antibody incubation time; in addition, if the background is very short, the background is deep It is possible that the closed non-specific protein in front of you is incomplete, and it is necessary to extend the blocking time; if the DAB coloration time is very long (such as more than ten minutes), positive staining may occur, which may indicate that your antibody concentration is too low or the incubation time is too short ( It is best to have a primary antibody of 4 degrees overnight; on the other hand, the closure time is too long.
12, counterstaining
The goal is to form a cell contour that better localizes the target protein, often counterstained with hematoxylin (nuclear dye). Note that the hematoxylin counterstaining time depends on the room temperature at the time, the old and new solutions, the location of the target antigen, etc. Generally, the cytoplasmic protein can be longer and the nucleoprotein is shorter in a few seconds to several minutes. However, this can be remedied if the dyeing is not ideal. That is, if the dyeing is deep, the differentiation time can be slightly longer; if the staining is shallow, it can be stained in hematoxylin.
Hydrochloric acid is a differentiation, and ammonia is returned to the orchid. The effect is different. After the film is counter-dyed, the water is shaken, then placed in hydrochloric acid alcohol for a few seconds (must be fast), then taken out of the water to be washed, and returned to the blue water in the ammonia water.
13, cover
For long-term storage, we generally use neutral gum to seal the film to avoid air bubbles. The method is to drop a piece of sealing liquid directly on the slide tissue, then hold the corner of the cover piece with one hand and the opposite side with the other hand. At the corner, the corner near the proximal end of the sealing liquid is lowered until it comes into contact with the liquid; when the liquid contact surface is found to be continuously dispersed, the other corner can be slowly lowered, so that no air bubbles are generally generated.
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