阅读说明：本技术 一种利用抗体芯片检测食管癌组织的方法 (Method for detecting esophageal cancer tissue by using antibody chip ) 是由 韩俊永 薛士杰 陈金烟 王坤 金静君 于 2019-11-11 设计创作，主要内容包括：本专利针对食管癌组织芯片的免疫组织化学常用技术进行改良,使的改良后芯片免疫组织化学技术更加节省抗体、背景更加清晰和显色效果更佳。(This patent improves to the immunohistochemistry common technique of esophagus cancer tissue chip, and the improvement back chip immunohistochemistry technique that makes saves the antibody more, the background is clearer and the color development effect is better.)

1. A method for detecting esophageal cancer tissues by using an antibody chip is characterized by comprising the following steps:

(1) putting the tissue chip into an oven, adjusting the temperature to 65 ℃, and baking wax for 12 hours;

(2) preparing a reagent: preparing 10 XPBS buffer solution, citric acid repair solution and EDTA repair solution;

(3) after the chips are baked, taking out the chips from the oven, putting the chips into a full-automatic dyeing machine, and dewaxing the chips;

dewaxing: xylene three cylinders, wherein the time of each cylinder is 4 minutes, 3 minutes and 3 minutes respectively; absolute ethyl alcohol is added into a cylinder for 3 minutes; two jars of 95v/v% alcohol, 2 minutes each time; 1 jar with 80v/v% alcohol for 2 minutes;

(4) taking out the slices from the dyeing machine, and washing with pure water for 3 times, each time for 1 minute; in the washing process, the citric acid repairing liquid is placed on an induction cooker to be heated;

(5) antigen retrieval: and C, citric acid high-pressure repair: after the citric acid repairing liquid is boiled, putting the slices into a pressure cooker, covering the pressure cooker cover, starting timing after the gas is continuously discharged, stopping heating after 1 minute and 40 seconds, placing the pressure cooker in room temperature, and opening the cover after cooling for 1 hour; EDTA heat repair: heating EDTA repair liquid to boil, putting the slices into the EDTA repair liquid, timing for 10 minutes, placing the EDTA repair liquid at room temperature, cooling for 1 hour, and opening a cover;

(6) preparing an endogenous peroxidase blocking agent;

(7) circling the tissue in a minimum range by using a 3ml immunohistochemical pen, and then dripping a blocking agent into the circle for 10 minutes;

(8) taking out the slices, washing with PBS buffer solution for 3 times, and washing for 1 minute;

(9) taking out the primary antibody from the refrigerator, and centrifuging for 30 seconds at 7200rpm/min in a centrifuge;

(10) taking out the primary antibody, and diluting the primary antibody by using DAKO antibody diluent according to the dilution;

(11) dripping primary antibody;

(12) throwing off redundant liquid, placing the chip in a wet box, dropwise adding primary antibody in the ring, and incubating for 3 hours at room temperature;

(13) washing the slices with PBS buffer solution for 3 times, once for 1 min;

(14) dripping an EnVision +/ HRP rabbit working solution or EnVision +/ HRP mouse working solution of a DAKO company until the solution can completely cover the tissue, and incubating for 30 minutes;

(15) after the time is up, washing the mixture for 3 times by using PBS buffer solution, and washing the mixture for 1 minute;

(16) taking out the DAB kit from the refrigerator, and preparing according to 1ml of DAB diluent and 1 drop of DAB chromogen;

(17) dripping diluted DAB on the sheet until the DAB can completely cover the tissue, developing for 5 minutes, and then washing for 5 minutes by tap water;

(18) dripping Haematoxylin (SIGMA) on the slices until the slices can completely cover the tissues for 40 seconds, immersing the slices in 0.25% hydrochloric acid alcohol for 2 seconds after the time comes, and washing the slices for 2 minutes by using tap water;

(19) putting the slices into a full-automatic dyeing machine for dehydration, and taking out the slices for sealing;

and (3) dehydrating: 1 jar with 75% alcohol for 5 minutes; 1 jar with 85% alcohol for 5 minutes; 1 jar with 95% alcohol for 5 minutes; two cylinders of absolute ethyl alcohol, each cylinder for 7 minutes; xylene was in two jars, 15 minutes each.

2. The method for detecting esophageal cancer tissue by using an antibody chip as claimed in claim 1, wherein the formulation of the 10 x PBS buffer in step (2) is: 80g NaCl, 2g KCl、15.35g Na₂HPO₄、2g KH₂PO₄Adding pure water to a constant volume of 1000 ml, wherein the pH is = 7.2;

the preparation method of the citric acid repair liquid comprises the following steps: 82mL of 0.1mol/L sodium citrate solution, 18mL of 0.1mol/L citric acid solution and 900 mL of pure water are placed in a pressure cooker;

the preparation method of the EDTA repair liquid comprises the following steps: 50 x EDTA antigen retrieval solution at pH =9.0 was diluted 50-fold with purified water.

3. The method for detecting esophageal cancer tissue by using an antibody chip according to claim 1, wherein the endogenous peroxidase blocker in the step (6) is prepared by a method comprising the following steps: 38.4ml of anhydrous methanol +12ml of 30v/v% H₂O₂+9.6ml of purified water.

4. The method of claim 1, wherein the PBS buffer is prepared by adding 10 XPBS buffer to 1 XPBS buffer in step (2) and adding Tween 0.05% of the total volume of the buffer to the 1 XPBS buffer.

5. The method for detecting esophageal cancer tissue by using an antibody chip of claim 1, wherein the 0.25% alcohol hydrochloride prepared in step (18) is prepared by the following steps: 400mL of 70v/v% ethanol +1mL of concentrated hydrochloric acid.

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting esophageal cancer tissues by using an antibody chip.

Background

China is the country with the most number of esophageal cancer, wherein the Taihang mountain area is the region with high esophageal cancer incidence. According to the evaluation result of the Chinese malignant tumor, the average new cases and the average death cases of the esophageal cancer in China are ranked in the first four places of malignant tumors, and a large number of researches for many years show that the esophageal cancer is influenced by genetic factors and environmental factors.

The prognosis of patients with esophageal cancer is closely related to the date of their findings, but more than 95% of patients who are clinically diagnosed with esophageal cancer for the first time are in the middle-and-advanced stages. The research shows that: the five-year survival rate for early esophageal cancer is approximately 90%, while the 5-year survival rate for patients in the middle and late stages is approximately 10%. Therefore, screening high risk groups of esophageal cancer and establishing esophageal cancer risk early warning are the key points for reducing the mortality rate of patients with esophageal cancer.

At present, the method for detecting esophageal cancer by using an antibody chip is one of common methods for detecting esophageal cancer tissues, but the existing antibody chip detection kit has the defects of insecure tissue adhesion, easiness in flaking off, unclear background, poor color development effect and large antibody using amount.

Disclosure of Invention

The invention aims to provide a method for detecting esophageal cancer tissues by using an antibody chip, compared with the prior art, the tissue adhesion is firmer and the esophageal cancer tissues are not easy to fall off; dewaxing is more thorough, and later antigen restoration and clear background presentation are facilitated; the usage amount of the primary antibody is saved, and the color development effect is better.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting esophageal cancer tissues by using an antibody chip comprises the following steps:

(1) putting the tissue chip into an oven, adjusting the temperature to 65 ℃, and baking wax for 12 hours;

(2) preparing a reagent: preparing 10 XPBS buffer solution, citric acid repair solution and EDTA repair solution;

(3) after the chips are baked, taking out the chips from the oven, putting the chips into a full-automatic dyeing machine, and dewaxing the chips;

dewaxing: xylene three cylinders, wherein the time of each cylinder is 4 minutes, 3 minutes and 3 minutes respectively; absolute ethyl alcohol is added into a cylinder for 3 minutes; two jars of 95v/v% alcohol, 2 minutes each time; 1 jar with 80v/v% alcohol for 2 minutes;

(4) taking out the slices from the dyeing machine, and washing with pure water for 3 times, each time for 1 minute; in the washing process, the citric acid repairing liquid is placed on an induction cooker to be heated;

(5) antigen retrieval: and C, citric acid high-pressure repair: after the citric acid repairing liquid is boiled, putting the slices into a pressure cooker, covering the pressure cooker cover, starting timing after the gas is continuously discharged, stopping heating after 1 minute and 40 seconds, placing the pressure cooker in room temperature, and opening the cover after cooling for 1 hour; EDTA heat repair: heating EDTA repair liquid to boil, putting the slices into the EDTA repair liquid, timing for 10 minutes, placing the EDTA repair liquid at room temperature, cooling for 1 hour, and opening a cover;

(6) preparing an endogenous peroxidase blocking agent;

(7) circling the tissue in a minimum range by using a 3ml immunohistochemical pen (Fuzhou Mixin), and then dropwise adding a blocking agent in the circle for 10 minutes;

(8) taking out the slices, washing with PBS buffer solution for 3 times, and washing for 1 minute;

(9) taking out the primary antibody from the refrigerator, and centrifuging for 30 seconds at 7200rpm/min in a centrifuge;

(10) taking out the primary antibody, and diluting the primary antibody by using DAKO antibody diluent according to the dilution;

(11) dripping primary antibody;

(12) throwing off redundant liquid, placing the chip in a wet box, dropwise adding primary antibody in the ring, and incubating for 3 hours at room temperature;

(13) washing the slices with PBS buffer solution for 3 times, once for 1 min;

(14) dripping an EnVision +/ HRP rabbit working solution or EnVision +/ HRP mouse working solution of a DAKO company until the solution can completely cover the tissue, and incubating for 30 minutes;

(15) after the time is up, washing the mixture for 3 times by using PBS buffer solution, and washing the mixture for 1 minute;

(16) taking out the DAB kit from the refrigerator, and preparing according to 1ml of DAB diluent and 1 drop of DAB chromogen; (from DAKO liquid DAB + substrate System)

(17) Dripping diluted DAB on the sheet until the DAB can completely cover the tissue, developing for 5 minutes, and then washing for 5 minutes by tap water;

(18) dripping Haematoxylin (SIGMA) on the slices until the slices can completely cover the tissues for 40 seconds, immersing the slices in 0.25% hydrochloric acid alcohol for 2 seconds after the time comes, and washing the slices for 2 minutes by using tap water;

(19) putting the slices into a full-automatic dyeing machine for dehydration, and taking out the slices for sealing;

and (3) dehydrating: 1 jar with 75% alcohol for 5 minutes; 1 jar with 85% alcohol for 5 minutes; 1 jar with 95% alcohol for 5 minutes; two cylinders of absolute ethyl alcohol, each cylinder for 7 minutes; xylene was in two jars, 15 minutes each.

The formula of the 10 XPBS buffer solution in the step (2) is as follows: 80g NaCl, 2g KCl, 15.35g Na₂HPO₄、2gKH₂PO₄Adding pure water to a constant volume of 1000 ml, wherein the pH is = 7.2;

the preparation method of the citric acid repair liquid comprises the following steps: 82mL of 0.1mol/L sodium citrate solution, 18mL of 0.1mol/L citric acid solution and 900 mL of pure water are placed in a pressure cooker;

the preparation method of the EDTA repair liquid comprises the following steps: 50 × EDTA antigen retrieval solution (new biotechnology development, fozhou) at pH =9.0 was diluted 50-fold with purified water.

The preparation method of the endogenous peroxidase blocker in the step (6) comprises the following steps: 38.4ml of anhydrous methanol +12ml of 30v/v% H₂O₂+9.6ml of purified water.

The preparation method of the PBS buffer solution comprises the steps of changing the 10 XPBS buffer solution to the 1 XPBS buffer solution in the step (2), and adding Tween reagent accounting for 0.05 percent of the total volume into the 1 XPBS buffer solution.

The preparation method of the 0.25% hydrochloric acid alcohol in the step (18) comprises the following steps: 400mL of 70v/v% ethanol +1mL of concentrated hydrochloric acid.

The invention has the advantages that:

(1) the tissue adhesion is firmer, and the sheet is not easy to fall off;

(2) dewaxing is more thorough, and later antigen restoration and clear background presentation are facilitated;

(3) the usage amount of the primary antibody is saved;

(4) the color development effect is better.

Drawings

FIG. 1 is a graph showing the contrast between the color development effect and the background sharpness in example 1 and comparative example 1.

Detailed Description