Intraoperative quick immunohistochemical frozen-section staining method

A frozen section and immunohistochemical technology, applied in the field of medical pathology detection, can solve problems such as degradation, autolysis and deformation, protein destruction, antigen damage, etc., and achieve the effects of increasing incubation temperature, improving accuracy, and speeding up reaction time.

Inactive Publication Date: 2019-11-05
李海南
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of immunohistochemical staining, it is generally necessary to preserve the original structure of tissues and cells as much as possible to fix cells, to avoid degradation, autolysis and deformation of tissues and cells, a

Method used

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Examples

Experimental program
Comparison scheme
Effect test

comparative example 1

[0033] (1) Take fresh tissue and place it on a cryostat, and slice it after freezing, with a thickness of 6 μm;

[0034] (2) Mount the frozen section on a glass slide and air dry for 30 seconds;

[0035] (3) Fix with 95% alcohol for 2 minutes;

[0036] (4) Rinse with PBS three times, each time for 30s;

[0037] (5) Add the primary antibody dropwise and incubate in a 37°C incubator for 5 minutes, then wash with PBS three times, each time for 10 seconds;

[0038] (6) Add polymer enhancer and incubate in a 37°C incubator for 5 minutes, then rinse with PBS three times, each time for 10 seconds;

[0039] (7) Add the secondary antibody dropwise and incubate in a 37°C incubator for 5 minutes, then wash with PBS three times, each time for 10 seconds;

[0040] (8) DAB reagent was added dropwise and stained at 37°C for 1 min, rinsed with double distilled water three times, each time for 5 s;

[0041] (9) Counterstain with hematoxylin staining solution for 15 seconds, return to blue ...

comparative example 2

[0043] (1) Take fresh tissue and place it on a cryostat, and slice it after freezing, with a thickness of 5 μm;

[0044] (2) Stick the frozen section on the glass slide coated with L-polylysine anti-off tablet;

[0045] (3) Fix the slices in acetone for 1 min, wash with PBS three times, each time for 10 s;

[0046] (4) Add 3% hydrogen peroxide dropwise, put it into the incubation box and cover it, place it in the microwave oven for 1 minute at low-grade microwave, and then wash it with PBS three times, 10 seconds each time;

[0047] (5) Serum was added dropwise, placed in the incubation box and covered, and placed in a microwave oven for 5 minutes at a low-grade microwave;

[0048] (6) Add the primary antibody dropwise, put it into the incubation box and cover it, place it in the microwave oven for 8 minutes at low-grade microwave, and rinse it with PBS three times, each time for 10 seconds;

[0049] (7) Add the secondary antibody dropwise, put it into the incubation box and...

Embodiment example 1

[0053] (1) Slice the frozen specimen in a cryostat with a thickness of 5um, stick it between 1 / 3 and 2 / 3 of the slide glass, and air dry for 30s;

[0054] (2) Add the primary antibody dropwise and incubate for 4 minutes in a 40°C incubator;

[0055] (3) wash with PBS three times, each time for 5s;

[0056] (4) Add the secondary antibody dropwise and incubate for 4 minutes in a 40°C incubator;

[0057] (5) wash with PBS three times, each time for 5s;

[0058] (6) DAB reagent is added dropwise, and the color development is 1mim;

[0059] (7) staining with hematoxylin staining solution for 5s;

[0060] (8) Rinse with running water for 10 seconds, and seal the slide with neutral gum.

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PUM

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Abstract

An intraoperative quick immunohistochemical frozen-section staining method comprises the following steps: placing an intraoperative fresh specimen in a freezing microtome for freezing and then slicing, wherein the section thickness is 5 microns, pasting a section on a glass slide, and carrying out air drying for 25-35 seconds; dropwise adding a primary antibody for incubation at 40 DEG C for 4 minutes; executing washing with PBS three times for 4-7 seconds each time; dropwise adding a secondary antibody for incubation at 40 DEG C for 4 minutes; executing washing with PBS three times for 4-7 seconds each time; dropwise adding a DAB reagent, and executing development for 1-2 minutes; executing staining with hematoxylin stain for 3-10 seconds. The staining process is not fixed, endogenous blocking is avoided, the incubation temperature is increased, the reaction time is shortened, the steps are omitted, the staining time is shortened, the total experiment time is greatly shortened, the whole process time needs about 12 minutes, the possibility of assisting the rapid frozen pathology diagnosis time is provided, the frozen diagnosis accuracy is improved, and the method has good repeatability and comparability with immunohistochemical results of paraffin.

Description

technical field [0001] The invention relates to the technical field of medical pathological detection, in particular to an immunohistochemical staining method for intraoperative frozen sections. Background technique [0002] Intraoperative frozen rapid pathology is to quickly give a preliminary pathological diagnosis (<30min) to a small part of the patient’s resected tissue under the microscope during the operation, and the preliminary pathological diagnosis will guide the doctor’s next surgical treatment plan, which has important clinical value. However, frozen diagnosis has certain limitations. Many tumors can’t be diagnosed only by intraoperative frozen staining technique, and the diagnosis can only be confirmed after postoperative paraffin specimen immunohistochemical examination. In this case, intraoperative rapid frozen pathological diagnosis is the Lost the clinical value of its specified surgical treatment plan. If reliable immunohistochemical staining results ca...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N1/42
CPCG01N1/30G01N1/42
Inventor 李海南
Owner 李海南
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