Application of TIGAR protein in plasma as molecular marker in preparation of kit for predicting and diagnosing liver cancer recurrence
A molecular marker and kit technology, applied in the field of biotechnology detection, can solve the problems of increasing patient time, increasing the number of waiting lists, increasing the risk of liver transplantation dissemination and recurrence, etc.
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Embodiment 1
[0039] Plasma ELISA assay. Methods as below:
[0040] (1) Plasma thawing: The fresh frozen plasma of the liver cancer group and the control group, which were stored at -80°C, were taken out, and placed in a 4°C ice box to thaw.
[0041] (2) Use human fructose-2,6-bisphosphatase TIGAR ELISA kit (Wuhan Yiaibo Technology Co., Ltd., number: E5848h), the sensitivity of the kit is 0.085ng / mL, and the detection range is 0.15 -10.0 ng / mL. Take the kit out of the -20°C refrigerator and allow the reagents inside to equilibrate to room temperature.
[0042] (3) Gradiently dilute the standard product: first add the lyophilized standard product to the sample diluent to dilute 1.0mL, dissolve and mix well, and then let it stand for 10min. At this time, the concentration of the solution is 10.0ng / mL. 5.0ng / mL, 2.5ng / mL, 1.25ng / mL, 0.62ng / mL, 0.31ng / mL and 0.15ng / mL, put them into marked EP tubes respectively, and use the sample diluent as 0ng / mL , and its stock solution is used as the hi...
Embodiment 2
[0064] A. Extraction of total protein
[0065] Tissue homogenate: take 100mg of cancer tissue and paracancerous tissue respectively from fresh frozen tissue, wash away the blood water with 1×PBS, blot the water with filter paper; cut the tissue on ice with sterile scissors, add to a 1.5ml centrifuge tube Put the prepared tissue lysate into the shredded tissue. Homogenized steel balls were soaked with 75% alcohol for 30 minutes, washed with pure water for 3 times, each time for 3 minutes; ddH 2 O soak for later use. Wipe the steel balls with filter paper and add them to the centrifuge tube containing tissue lysate and tissue. Turn on the homogenizer 20 minutes in advance. After the internal temperature of the homogenizer drops below 4°C, put it into the prepared centrifuge tube and set up the frozen homogenizer. The parameters are 45Hz, 60s, 5 cycles, and stand still for 10min after the end. Take the centrifuge tube out of the homogenizer and put it into the low-temperature ...
Embodiment 3
[0105] The fixed and dehydrated tissue samples were divided into two parts. One part was processed according to the procedures of transparency, wax dipping and embedding. The parameters of the paraffin microtome were set at 4 μm for continuous sectioning. Adhesive glass slides were used to paste the tissues, and the cut paraffin sections were placed for 4 Store at ℃; another copy of OCT was embedded, and the parameters of the cryostat were set at 4 μm for continuous sectioning, and the slides were pasted to paste the tissue, and the cut frozen sections were stored in a -80°C refrigerator.
[0106] A. Hematoxylin-eosin staining (HE staining)
[0107] 1. Paraffin sections are baked: set the temperature at 60°C for 2-2.5 hours. 2. After the paraffin is melted, carry out the dewaxing hydration step. First, prepare 2 parts of xylene, 2 parts of absolute ethanol, 1 part of 95%, 80%, 75% and 50% ethanol, 1 part of distilled water, and each of the above liquids 250-350ml (subject to ...
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