Some lessons: 1. For specimens fixed with multiple nails, if they are to be stored for a long time, they must be dehydrated and then frozen. This method is more suitable for the IHC bleaching method. Correct operation: multi-perfusion fixation-post-fixation of multi-formaldehyde or formaldehyde-sucrose gradient dehydration-preservation at minus 70 degrees-frozen section error operation: multi-perfusion fixation-post-fixation of multi-formaldehyde or formaldehyde-minus 70 degrees Preservation—sucrose gradient dehydration—frozen sections, as a result, a large number of ice crystals will form. 2. If you can confirm the use of paraffin sections and use the patch method for the next step of IHC, dehydrate and embed paraffin as soon as possible after fixation of polymethyl or formaldehyde. Key points and operating techniques: 1. Fixation: It is best to use 4% paraformaldehyde fixative. For frozen sections, formaldehyde fixation is sometimes better than frozen acetone; but for different tissues and antigens, different fixing solutions can be used. Sometimes the commercially available antibody will have a suitable and recommended fixative solution. Please pay attention to the instructions before purchasing. Bouin S fixative: 750ml of saturated picric acid, 250ml of formaldehyde, 50ml of glacial acetic acid. It has strong penetrating power to tissues, good fixation and complete structure. Not suitable for long-term preservation of tissue specimens. PLP solution: namely sodium periodate-lysine-paraformaldehyde, suitable for fixing paraffin sections. It is suitable for carbohydrate-rich tissues and has good preservation of ultrastructure and antigenicity of many antigens. 2. The tissue is dehydrated and transparent: the time should not be too long, otherwise it will be easily broken and incomplete when sliced. 3. Spreading during sectioning: Some tissues are difficult to spread in water after sectioning, then a few drops of ethanol can be added to the water appropriately. 4. Baking slices: 60 ℃ for 30 minutes or 37 ℃ overnight, the temperature is too high or the time is too long, the antigen is easy to be lost. 5. Storage of wax blocks and slices: it is best to store at 4 ℃ Off-chip problem: Poly-L-Lysine (poly-lysine) is one of the most commonly used anti-off tablets in immunohistochemical staining work. 6ml of poly-lysine solution can be diluted 1:10 into 60ml The working fluid is suitable for anti-strip processing that requires enzyme digestion, microwave, high temperature and high pressure. If not, double-processing (APES and Poly-L-Lysine) slices can be used. In the case where the above two conditions are not feasible, the following methods can be used: slices are soaked in APES 1:50 acetone solution for 3 minutes before dewaxing, and dried to proceed to the next step. 7. Inactivation of endogenous enzymes: HRP system: 3% hydrogen peroxide inactivation; AP system: 3% HAc inactivation. 8. Antigen exposure: For the immunohistochemistry experiment of paraffin sections, high temperature heating of antigen repair must be used, which will help to expose the antigenic determinants, thereby increasing the intensity of immunohistochemical staining (for the optimal repair solution of different antibodies, please refer to the antibody manual) ). For different tissues, different antigens, and different antibodies, the method used should be different. It can be heat repaired, trypsinized, and neither repaired nor digested. Collagen can also be digested with pepsin. 9. Blocking: When the goat serum is blocked and the non-specific staining is still strong, the blocking time can be extended or blocked with concentrated serum. Antibody dilution: follow the principle of "ready-to-use". Antibodies diluted in PBS must be used on the same day. 11. High background: Under suitable conditions such as antibody concentration, reaction time, reaction temperature, etc., if the background is still high, you can use PBS containing 1 ‰ Tween20, especially before washing. 12. Return to blue: After counterstaining with hematoxylin, use alkaline buffer (such as PBS) or Na2HPO4 saturated solution to return to blue. 13. Color rendering: Be sure to observe under the microscope, pay attention to control the background. Source: Biotechnology Forum
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