1. It is recommended to use fresh tissue for the preparation of frozen sections. Otherwise, the internal structure of the tissue cells will be destroyed, and the antigen will be easily dispersed. Choose a clean and sharp blade, the tissue must be frozen moderately, etc., to prevent severe cracking and peeling. 2, tissue section fixed sliced ​​air-dried immediately fixed with ice acetone and other fixed solution for 5-10 min, especially for long-term preservation of the white film, must be fixed and properly stored in time. 3. Serum blockade To prevent the binding of endogenous non-specific protein antigens, it is necessary to block the serum (consistent with the source of the secondary antibody) before the primary antibody is incubated, thereby reducing the background coloration. The time of serum blocking can be adjusted, usually 10-30 min. 4. 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. 5, secondary antibody incubation conditions The second resistance is generally room temperature or 37 degrees 30 min-1 h, 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 remaining after the storage time is extended. It is necessary to pay attention to the small package during preparation and appropriate centrifugation. 6. The purpose of counterstaining is to form a cell contour to better localize the target protein. DAPI counterstaining is commonly used. 7. For long-term storage, we usually use buffer 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 in one hand and the opposite corner of the cover, lowering the corner near the proximal end of the sealant until 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 (extended time and increase the number of times). I usually wash 3 min*3 times before the primary antibody incubation. The wash after incubation with primary antibody 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) If you have a photo, if you can't take photos in time, you should seal the film and seal it with nail polish to keep it 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 the time every time 1~ 2 h is suitable, more than 90 min, the luminous intensity of the ultra-high pressure mercury lamp gradually decreases, and the fluorescence is weakened; after the ultraviolet light is irradiated for 3 to 5 minutes, the fluorescence is also obviously weakened or faded; the fluorescence of the excitation light is attenuated for a long time. And quenching phenomenon; so the maximum should not exceed 2~3 h; the fluorescence microscope light source has a limited life, 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 equipped 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.
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