First, the key link of enzyme immunohistochemistry
1. Specimen fixation: The purpose of fixation is to prevent the specimen from falling off the slide; 2 to remove the lipid that prevents antigen-antibody binding, so that the antigen-antibody conjugate is easy to obtain good staining results; 3 the fixed specimen is easy to preserve.
2, dehydration, paraffin embedding and tableting: dehydration with gradient ethanol (low to high) fully dehydrated, the tissue should be completely waxed, the blade should be clean and sharp. Otherwise, it is easy to split and peel off.
3. Dewaxing and hydration: This is because the reagents such as the latter can fully bind to the antigen in the 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 3-4h. 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: due to the partial immobilization of formaldehyde in the process of formaldehyde or paraformaldehyde, the cross-linking between proteins and the blocking of aldehyde groups occur, thus losing antigenicity; through antigen repair, the intracellular epitopes are re-established Exposure increases antigen detection 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 30min (as long as you think the temperature of the repair solution reaches room temperature).
5. Cell Permeability: 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, TritonX-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 be smoothly performed. Enter the intracellular and bind 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 peroxidation must be used. Hydrogen and avidin are inactivated. 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 10~30min; use hydrogen peroxide to configure hydrogen peroxide than double distilled water Or PBS may be good at 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 blocking: the remaining sites on the tissue section can be non-specifically combined with the primary antibody, resulting in false positive results; the closed serum is generally the same source as the secondary antibody, the serum of the animal's own antibodies, pre-energy and tissue The sites with cross-reactivity are 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 10-30min.
8. Primary and secondary antibody concentrations and incubation times: 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 1-2h, and 4 degrees overnight and taken out from the refrigerator After 37 degrees of rewarming 45min. Specific conditions have to explore. Secondary antibody incubation conditions: the secondary antibody is generally room temperature or 37 degrees 30min-1h, 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 PBS components, special antibody dilutions, plus sodium azide preservatives, BSA stabilizers and other components, Multiple recycling of antibodies is preferred. 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 false negative results eventually appeared.
10, slice cleaning (dipping, rinsing and rinsing): In order to prevent non-specific staining caused by residual primary antibody, secondary antibody and other reagents, it is especially important to strengthen the cleaning (extended time and increase the number of times), I usually incubate in primary antibody The previous cleaning was 3 min*3 times, and the cleaning after the primary antibody incubation was 5 times*5 min.
Note: (1) Wash separately to prevent contamination caused by 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 0.01M at 7.4-7.6. (Neutral and weak sputum conditions (PH7) -8) Conducive to the formation of immune complexes, while acidic conditions facilitate 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 you need 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 purpose is to form a cell contour to better target 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-minutes. However, this can be remedy if the dyeing is not ideal. That is, the dyeing depth is longer when the differentiation time is slightly longer; if the staining is shallow, it can be stained in hematoxylin.
Hydrochloric acid alcohol is differentiated, and ammonia water is returned to the orchid. Different effects. After the film is over-dyed, the water is shaken, then placed in hydrochloric acid alcohol for a few seconds (must be fast), then taken out of the water and shuffled, and then returned to the blue water in the ammonia water.
13. Covering: 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, and then hold the corner of the cover piece with one hand, and another With the opposite corner of the handle, the corner near the proximal end of the seal liquid is lowered until it comes into contact with the liquid; when the liquid contact surface is found to be constantly diffusing, the other corner can be slowly lowered, so that bubbles are generally not generated.
Second, the important link in the immunofluorescence method
1. Preparation of frozen sections: It is recommended to use fresh tissue, otherwise the internal structure of the tissue cells will be destroyed, and the antigen will be easily dispersed. Use clean and sharp blades, tissue must be moderately frozen, etc., to prevent severe cracking and peeling.
2, tissue section fixation: cut into a good air immediately after fixing with a fixed solution of ice acetone for 5-10min, especially for a long time to save the white film, must be fixed and properly stored in time.
3, serum blocking: In order to prevent the binding of endogenous non-specific protein antigens, it is necessary to use serum (consistent with the source of secondary antibodies) to block before the primary antibody is incubated, and to reduce the background coloration. The time of serum blocking can be adjusted, generally 10-30min.
4, primary antibody incubation conditions: the most important in the 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 1-2h, and 4 degrees overnight and taken out from the refrigerator After 37 degrees of rewarming 45min. Specific conditions have to explore. Immunohistochemistry experiment
5, secondary antibody incubation conditions: the second resistance is generally room temperature or 37 degrees 30min-1h, the specific time needs to explore, and the concentration generally has working fluid, if it is concentrated solution to explore the concentration, remember to avoid light reaction. However, in immunofluorescence, 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. Finally, the fluorescein-labeled secondary antibody may have a large amount of free fluorescein residue after prolonged storage time, and it is necessary to pay attention to the small package during preparation and appropriate centrifugation.
6. Counterstaining: The purpose is to form a cell contour to better localize the target protein. DAPI counterstaining is commonly used.
7. Mounting: For long-term storage, we usually use buffering glycerin and other sealing sheets, in addition to special anti-fluorescence extraction and sealing liquid. Avoid creating bubbles by dropping a drop of sealant directly onto the slide tissue, then holding the corner of the cover slip with one hand and the opposite corner of the cover with the other hand, and lowering the corner near the proximal end of the sealant until the corner is lowered. When it comes into contact with the liquid; when it is found that the liquid contact surface is constantly diffusing, the other corner can be slowly lowered, so that bubbles are generally not generated.
8. Slice cleaning: 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 (prolonging the time and increasing the number of times). I usually wash the primary antibody before 3 minutes*3 times. However, the cleaning after incubation with the primary antibody was 5 times *5 min. Note (1) Washing separately to prevent contamination by 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 0.01M at 7.4-7.6. (Neutral and weak sputum conditions (PH7) -8) Conducive to the formation of immune complexes, while acidic conditions facilitate decomposition; low ionic strength favors the formation of immune complexes, while high ionic strength facilitates decomposition).
9, photo: If you have the conditions, it is best to take pictures immediately, if you can not take photos in time, also seal the film and seal with nail polish, keep away from light and humidity. Use fluorescence microscope to strictly follow the requirements of the fluorescence microscope factory instructions, do not change the program arbitrarily; check in the dark room; prevent ultraviolet rays from damaging the eyes, wear protective glasses when adjusting the light source; check time every time 1~ 2h is suitable, more than 90min, the luminous intensity of the ultra-high pressure mercury lamp gradually decreases, and the fluorescence is weakened; after the laser is irradiated by ultraviolet light for 3~5min, the fluorescence is also obviously weakened or faded; when the excitation light is irradiated for a long time, the fluorescence decay and quenching will occur. Phenomenon; so the maximum should not exceed 2~3h; the fluorescence microscope light source has a limited life, and the specimen should be intensively checked to save time and protect the light source. When it is hot, the fan should be cooled and cooled down. The new lamp should be recorded from the beginning. When the light is turned off and you want to re-enable it, it needs to be fully cooled before it can ignite. Avoid igniting the light source several times a day. Turn off the mercury lamp at least 15-30 minutes after opening; observe the specimen immediately after staining, and the fluorescence will gradually weaken due to the long time. If the specimen is stored in a polyethylene plastic bag at 4 ° C, the fluorescence decay time can be delayed and the sealing agent can be prevented from evaporating. The carrier such as the slide glass must have uniform thickness and no obvious autofluorescence. If oil mirror is used, It is necessary to ensure that the mirror oil is non-fluorescent oil; the power supply is best installed with a voltage regulator, otherwise the voltage instability will not only reduce the life of the mercury lamp, but also affect the effect of the microscopic inspection.
Urinalysis strip is a strip that tests for bilirubin, urobiligen, ketone bodies, ascorbic acid, glucose, proteins (albumin), blood cells, PH, etc.
1. Dip the test paper into the fully stirred fresh urine and take it out in time;
2. Put the test paper on the edge of the container to drop excess urine or put it on a paper towel to suck away excess liquid to reduce interference.
3. Judgment:
(1) in the time specified on the color code and the standard color code for color, judgment and read the results;
(2) For the operation of the instrument, please refer to the manual of the Urine Analyzer used.
4. When the test paper is visually tested, the reaction results are valid for interpretation within 60 seconds, and the interpretation beyond 60 seconds is invalid.
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