Experimental reagent
1. 10% neutral formalin (pH 6.8 ~ 7.1) formaldehyde 10ml distilled water 90ml sodium acetate 2g
2. Acid phosphatase solution
Distilled water 90ml 0.2mol/L acetate buffer (pH4.6) 12ml 5% lead nitrate 2ml
3. 2% β-glycerol phosphate 4ml
Distilled water and acetic acid buffer first, then divided into two equal parts, one part added with lead nitrate solution, the other part added with sodium glyceryl phosphate solution, and then slowly mixed the two, while mixing and mixing The pH value is less than pH 5.0, and a small amount of acetic acid can be adjusted. This working solution is preferably formulated before use and cannot be stored. After the solution, the liquid should be transparent and free of flocculent suspended matter and precipitate, otherwise the experimental results will be seriously affected. Shanghai Chuangsai Technology provides 9001-77-8, acid phosphatase Acid PhosphataseBR, 10u/mg, liquid, commodity number: D23-RM1170-500U, price 6080 yuan.
4. 1% ammonium sulfide solution
Sulfide amine 1ml distilled water 9ml
5. Giemsa Dye (1:30)
Giemsa stock solution 3 drops of phosphate buffer (pH 6.8) 5ml
6. 6% starch broth
Beef Cream 0.3g Peptone 1.0g Sodium Chloride 0.5g Distilled Water 100ml
Autoclave 9.9 × 104pa (15 lbs) for 20min, then add soluble starch 6g, water bath, promote dissolution and store in the refrigerator.
Experimental procedure
1. Take one of the mice and inject 1 ml of 6% starch broth daily for 3 days.
2. 3 to 4 hours after the third day of injection, 1 ml of normal saline was injected into the abdominal cavity. After 3 minutes, 0.1 to 0.2 ml of peritoneal fluid was taken at the original injection site. Please note that the injection site and depth of the injection should be consistent. Otherwise, the peritoneal fluid may not be extracted. Shanghai Chuangsai Technology provides 9001-77-8, acid phosphatase Acid PhosphataseBR, 0.5-3u/mg, powder, commodity number: D23-RM1170-100mg, price 736 yuan.
3. Drop the abdominal cavity on the pre-cooled slides, 1 to 2 drops per piece, smear, insert the slides vertically into the slide holder, and quickly place them in the refrigerator (4 ° C) to allow the cells to spread themselves.
4. After 30 minutes, slide the slide into 10% neutral formalin and fix in the refrigerator for 30 min at 4 °C.
5. Rinse the tap water for 5 minutes and dry the water.
6. Transfer to acid phosphatase solution and treat at 37 ° C for 30 min.
7. Rinse the tap water for a while.
8. 1% ammonium sulfide treatment for 3 to 5 minutes.
9. Rinse with tap water and dry.
10. Stain with 1:30 Giemsa stain for 15 min.
11. Tap the water to wash the dye solution, temporarily seal it with phosphate buffer, and check it.
12. Control experiment:
The peritoneal fluid smear of the third step was treated in a 50 ° C incubator for 30 min to inactivate the enzyme, and the same experiment was carried out for 6 to 11 steps.
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