There are many types of fixatives commonly used, and the correct choice of fixative depends on the nature of the antigen being studied and the nature of the antibody used. Fixatives can be divided into two categories: organic solvents and crosslinkers. Organic solvents: such as ethanol and acetone can remove lipids to dehydrate cells and precipitate proteins on the cell structure. Crosslinking agents: For example, paraformaldehyde generally bridges biomolecules through free amino groups to form a cross-linked antigenic network. Many people find that it is better to fix cells with cross-linking agents, especially when doing immunofluorescence, of course, there is no fixed rule. However, cells fixed with paraformaldehyde alone do not allow the antibody to enter the cell. Therefore, the specimen must be permeabilized with a non-ionic detergent after the specimen is fixed. method one 1) 900 ml dd H20 500 ul 1N NaOH 2) Heat to 65-70°C 3) While stirring, add 40 g paraformaldehyde 4) Continue heating and stirring until paraformaldehyde is dissolved 5) Let cool to room temperature 6) Add 100 ml of 10X PBS 7) pH to 7.3 with HCl 8) Sterile with filter 9) Store at 4°C, protected from light Method Two 1) 40 g of PFA (paraformaldehyde) was weighed and dissolved in a glass vessel (beaker or flask) containing 500 ml of DEPC water, and heating was continued. Shanghai Chuangsai Technology provides paraformaldehyde, Paraformaldehyde, commodity number: B11000407-25g, price 41.3 yuan. 2) Magnetically stir to 60-65 ° C, about 1-2 hours to dissolve, into a milky white suspension. Shanghai Chuangsai Technology provides E22-MYP11-2A intelligent constant temperature magnetic stirrer, product number: E22-MYP11-2A, inquiry. 3) Adjust the pH to 7.0 with 1.0 mmol/L NaOH to make it clear (drop), then add about 500 ml of 2×PBS and mix well (in an ice bath or a cold water bath). Shanghai Chuangsai Technology provides PBS buffer, product number: D16-1027378-2ml, the price is 23 yuan. 4) Detect PH, filter to volume to 1000ml, room temperature or 4 ° C (preserved for use), freshly prepared before use. note: 1. When working, it should be operated under ventilated conditions, and avoid contact with skin and inhalation (wearing masks and gloves). Because PFA has strong toxicity, it has a stimulating effect on mucous membranes and skin. 2. When heating, the temperature should not be too high, usually 60-65 °C. Otherwise, the degradation of PFA is invalid, and the prepared PFA can be effective for a little time, but the liquid is too long, the fixing effect is lowered, and it is freshly prepared before application. In addition to formulating PFA with DEPC water, it can also be prepared with sterile distilled water or DEPC-treated 0.01-0.1%/L PBS. The method and main items are the same as above. Shanghai Chuangsai Technology provides diethyl pyrocarbonate, DEPC (Diethylpyrocarbonate), 1609-47-8, commodity number: B11000132-5ml, and the price is 64.5 yuan. CANSPEC quality provides excellent performance, its excellent quality to meet the various needs of your different experiments, spot, product details, scientific instruments, laboratory supplies, Shanghai Chuangsai, mass products special promotions, welcome to inquire! Stainless Steel Commercial Steamer Stainless Steel Steamer,Stainless Steel Food Steamer ,Kitchen Steel Steamer,Stainless Steel Steamer Cooker Jiangmen T2grand Stainless Steel Kitchenware Factory , https://www.yapamit.com
4% paraformaldehyde preparation method