I ¨C Preparation of fresh paraformaldehyde (PFA)
This recipe yield 250 mL of a 20% PFA solution in 10 mM sodium phosphate buffer (Ph 7.2) with 500 mM NaCl. Bottles, filter apparatus, etc. used for preparing PFA should never be used for live samples. 1. Turn on a 60?C water bath in advance.
2. Pour 150 mL of deionized water into a 250 mL screw cap polycarbonate bottle. Add 50 g of PFA.
3. Fasten the cap securely and shake well to disperse the PFA powder. 4. Add 3 mL of 1 N, NaOH.
5. Warm to 60?C for 20-25 minutes, shaking the bottle every 5 minutes. Most but not all the PFA will dissolve in this time. It will still be quite cloudy. The last step is 0.2 m filtration, which will clear it up. (
Note: If you cook the PFA too
long at 60?C it will become acidic, making it difficult to adjust the pH.) 6. Add 3 mL of 1 N HCl
7. Add 25 mL of 100 mM sodium phosphate buffer, pH 7.2.
8. Adjust pH to 7 using pH paper. This will take approximately 400 l of 1 N, NaOH.
9. Adjust the final volume to 250 mL with deionized water using the graduations on the polycarbonate bottle.
10. Filter using a 0.22 m Millipore type GS filter (47 mm) using a designated PFA side arm flask, frit and cup. There may be a substantial layer of PFA on the filter.
11. Transfer the filtrate to a clean 250 mL polycarbonate bottle. Label with the date, concentration (20% PFA) and your initials.
12. Store at 4?C. Fresh PFA is good for 2-3 days if kept in the refrigerator. PFA can be stored frozen for longer periods of time. 多聚甲醛的配制
首先将水浴锅温度调至60度
于250ml旋口塑料瓶中加入150ml去离子水，并加入50g多聚甲醛 盖好盖，充分摇匀使多聚甲醛粉末溶解 加3ml 1N 的氢氧化钠
60度温浴20_25分钟，期间第隔5 分钟摇一次瓶，促进多聚甲醛的溶解，在这一过程中并非所有的多聚甲醛都会溶解，在最后用0.2微米的滤膜过滤将其清除 温浴时间不可太长，否则多聚甲醛会变酸，这将很难调pH值 加3ml 1N的盐酸
加25ml的100mM pH值为7.2的磷酸盐缓冲液(分别配制成100mM的母液) 磷酸二氢钠：磷酸氢二钠=28:72(ml)
调pH值至7,这一步将大约消耗400 l 1N的氢氧化钠 用去离子水调终体积至250ml , 0.22微米滤膜过滤
将滤液装入干净的250ml的塑料瓶，标明日期，浓度
4度保存，新制的PFA在冰箱中可保存2-3天，冷冻可保存很长时间