First, the preparation of homogenization medium:
Generally, pH 7.4, 0.01 mol/L Tris-HCl, 1 mmol/L EDTA-2Na, 0.01 mol/L sucrose, 0.8% sodium chloride solution or 0.86% physiological saline is used as the homogenization medium.
Second, the preparation of tissue homogenate
1, take the tissue block (0.1g ~ 0.2g) at least 2 ~ 5mg rinsed in ice cold saline, remove the blood, filter paper dried, accurately weighed, placed in a 5ml homogenate tube.
2, according to the weight (g): volume (ml) = 1:9 ratio of 9 times the volume of homogenization medium (pH 7.4, 0.01mol / L Tris-HCl, 1mmol / L EDTA-2Na, 0.01mol / L Sucrose, 0.8% sodium chloride solution) or 0.86% physiological saline in the homogenate tube, and the tissue block was cut as soon as possible with an ophthalmic small scissors under ice water bath conditions.
3, there are a variety of ways of homogenization: manual homogenization, machine homogenization, ultrasonic pulverization.
① Manual homogenization: insert the lower end into the vessel containing the mixture of ice water and water in the left hand. Insert the mast into the casing vertically by the right hand, rotate it up and down for dozens of times (6-8 minutes), fully grind and make. Into 10% homogenate.
② Machine homogenization: 10% tissue homogenate is prepared by grinding with 10,000 to 15,000 rpm of tissue mincer. It can also be prepared by internal cutting homogenizer (homogenization time 10 sec/time, gap 30 sec, continuous 3 to 5 times, in ice water), skin, muscle tissue, etc. can extend the homogenization time.
③ ultrasonic pulverization: pulverization with ultrasonic pulverizer, sonicep150 type ultrasonic generator can be used to ultrasonically process the amplitude of 14 micron for 30 seconds to break the cells. It can also be used with domestic ultrasonic generator, with 400 amps, 5 seconds/time, and gap 10 seconds. ~5 times.
Microscopic examination: Take a small amount of tissue homogenate for smear (direct smear, staining can be), observe whether the cells are broken under the microscope, if not, extend the homogenization time.
4. Prepare the prepared 10% homogenate by using a common centrifuge or a low-temperature low-speed centrifuge at 2,500 rpm, centrifuge for 10 to 15 minutes, and take the supernatant for determination.
Third, the sample is saved:
Animal tissue samples are not measured for a while, and can be cryopreserved immediately. The lower the temperature, the better. If the temperature is not repeated in the middle, it can be stored for three months below -20 °C, and can be stored for six months below -70 °C.
The prepared homogenate is not recommended to be frozen. It is best to measure it on the same day. If the time is too long, the enzyme activity will decrease. Some indicators can be stored for 4 to 5 days at 4 °C (such as SOD for 2 to 3 days, MDA) Can store 3 to 5, total protein can be stored for 5 to 7 days, etc.)