CK20 Product Manual

Cytokeratin 20 Instruction Manual

Manual No.:MyR1-CK20253

(This antibody is for research use only, not for clinical diagnosis)

【Product Name】

【Product No. and Packaging Specifications】

【Intended Use】

This antibody reagent is suitable forimmunohistochemical stainingof formalin-fixed, paraffin-embedded human tissue sections, for identifying the expression of Cytokeratin 20 protein in tissues.

Cytokeratin 20 (CK20) has a molecular weight of 46kDa and mainly labels gastrointestinal epithelium, transitional epithelium and Merkel cells. CK20 is mainly used for the identification of gastrointestinal adenocarcinoma, cholangiocarcinoma, pancreatic cancer, transitional cell carcinoma and ovarian mucinous tumors. It is not expressed in breast cancer, lung cancer, endometrial cancer, ovarian non-mucinous tumors and small cell carcinoma. It is often used in combination with CK7 to determine the tissue origin of adenocarcinoma.

(Note:Interpretation of immunohistochemical staining results should be combined with pathological morphology and other data, and the staining results are not used as diagnostic indicators.)

【Main Components】

Monoclonal rabbit anti-human Cytokeratin 20 protein antibody, Clone No.: MyR1-CK20, Immunoglobulin Isotype: IgG. This product is tissue culture supernatant, containing 0.05 mol/L Tris-HCl (pH 7.2), 0.015 mol/L Sodium Azide (NaN₃) and protein stabilizers.

【Storage Conditions and Validity Period】

The reagent should be stored in the dark at 2-8℃ (not lower than 0℃). Return it to the 2-8℃ refrigerator immediately after each use. The validity period is shown on the outer package of the product (Note:Improper storage of the reagent as above may affect its usage effect).

【Principle of Immunohistochemical Staining】

In immunology, antigen and antibody molecules specifically bind due to structural complementarity and mutual affinity. Based on this principle, the antibody will bind to specific antigens/proteins in the tested human tissues and cells. Then, the enzyme chromogen labeling the antibody is developed through redox chemical reactions, thereby showing the antigen distribution in tissues and cells.

First, this antibody specifically binds to the antigen on the tested tissue; second, the enzyme-labeled anti-mouse/rabbit IgG polymer recognizes the antibody that has bound to the antigen; third, the chromogenic substrate is added, and the horseradish peroxidase on the polymer can catalyze the decomposition of H₂O₂ in the DAB chromogenic solution, oxidizing benzidine to benzidine imine, resulting in yellow or brown-yellow staining at the antigen sites in the tissue sections; finally, the samples are counterstained and mounted. The presence and expression of the antigen on the tissue sections are inferred by observing the staining under a microscope.

【Sample Requirements】

Fresh biopsy or surgical samples are fixed with 10% neutral formalin solution for 6-24 hours, and made into wax blocks by sampling, dehydration and paraffin embedding according to technical specifications. The wax blocks can be stored at room temperature for a valid period of 5 years.

Section the specimen wax blocks with a thickness of 3-5μm, adhere them to anti-drop slides, and bake the slides in a constant temperature incubator at 58-60℃ for 1 hour for staining. Unused unstained slides are stored at room temperature (15-25℃) (preferably with anti-oxidation treatment). For a good reproduction of antigen distribution in tissue sections, it is recommended to complete the immunohistochemical staining within 7 days.

【Staining Method】

The following are the recommended configuration and steps for manual immunohistochemistry. If an automated immunohistochemistry instrument is used for staining, adjustments should be made according to different instruments and chromogenic kits. A complete immunohistochemical experiment should set up positive tissue control, negative tissue control and blank (or negative) reagent control. For the evaluation and verification of experimental results, please refer to【Quality Control】and【Interpretation of Staining Results】.

1） Required Instruments and Equipment:

Induction cooker, stainless steel pot, timer, incubation wet box, staining rack, staining jar, coverslip, optical microscope (40×-200×), wash bottle, absorbent paper, pipette, etc.

2） Reagent and Material Preparation:

The working solution of this antibody reagent does not need to be diluted before use. If it is a concentrate, it is recommended to dilute it into a working solution with PBS buffer or antibody diluent at a ratio of 1:100-1:200 before use. For the preparation of PBS buffer, antigen retrieval solution, secondary antibody, DAB chromogenic solution and other reagents, please refer to the respective product instruction manuals.

Other materials required for the experiment are as follows:

PBS buffer (pH7.2-7.4), Tris-EDTA antigen retrieval solution (pH9.0), endogenous peroxidase blocker, HRP-labeled anti-mouse/rabbit IgG polymer, DAB chromogenic solution, hematoxylin staining solution, blank (or negative) control reagent, positive and negative control tissue sections, xylene or xylene substitute, ethanol (anhydrous, 95%, 85%), distilled water or deionized water, mounting medium (such as neutral balsam), anti-drop slides, coverslips, etc.

3） Experimental Temperature Conditions: Room temperature 18℃-28℃.

4） Experimental Steps:

A、Deparaffinization and Hydration

(1) Immerse the tissue sections in 2 fresh jars of xylene successively for 10 minutes each;

(2) Immerse the tissue sections in anhydrous ethanol, 95% ethanol and 85% ethanol successively for 5 minutes each;

(3) Rinse gently with distilled water for 1 minute, and soak in distilled water for retrieval after rinsing.

B、Antigen Retrieval (Pressure Cooker Retrieval)

(1) Put the prepared Tris-EDTA antigen retrieval solution (pH9.0) into a pressure cooker, place the deparaffinized and hydrated tissue sections on a high-temperature resistant plastic slide rack, put them into the retrieval solution, and ensure that the tissue sections are completely immersed in the retrieval solution;

(2) Cover the pressure cooker, fasten the pressure valve, heat on high fire (1600W/210℃) until steam is emitted, start timing for 2 minutes from the emission of steam and adjust to medium fire (800W/130℃). After the timing ends, turn off the induction cooker, move the pressure cooker away from the heat source steadily, and cool naturally for 5 minutes;

(3) Move the pressure cooker steadily to the sink, turn on running water to pour on the pot cover to cool it down until the automatic lock core falls into place, turn off the running water, remove the pressure limiting valve, and open the pot cover carefully (note steam scald). Then slowly inject running water into the pot (avoid direct flushing of running water to the tissue sections);

(4) When the temperature drops below 40℃ (the water temperature is not hot to the touch), take out the slide rack and immerse it in distilled water quickly (avoid drying of sections), rinse gently with distilled water for 3 minutes × 2 times, and soak in distilled water for blocking after rinsing.

(Note: Tissue sections are prone to drying after retrieval. In the subsequent operations, attention should be paid to preventing the tissue sections from drying when "shaking off excess liquid from tissue sections", otherwise the staining effect will be affected.)

C、Blocking of Endogenous Peroxidase

(1) Shake off excess liquid from tissue sections, place the slides in peroxidase blocker (3% H₂O₂) and soak for 10 minutes;

(2) After blocking, rinse gently with distilled water for 3 minutes × 2 times, and soak in distilled water for circle drawing after rinsing.

D、Circle Drawing

Shake off or carefully wipe off excess liquid around the tissue sections, draw a circle around the tissue with an immunohistochemical oil pen at a position 2~3mm from the tissue edge. After ensuring the entire circle encloses all the tissue, rinse gently with distilled water for 3 minutes × 2 times, rinse off excess oil, and soak in PBS buffer for primary antibody incubation.

E、Adding Primary Antibody or Control Reagent

(1) Shake off excess liquid from tissue sections, add the primary antibody working solution to completely cover the tissue after the circle drawn by the oil pen is fully visible, and incubate at room temperature in a wet box for 30 minutes;

(2) After primary antibody incubation, rinse with PBS buffer for 2~3 times, then transfer to a PBS jar and soak for 2 minutes × 3 times.

(Note: During the experiment, do not rinse the tissue surface directly when rinsing tissue sections with PBS buffer, otherwise section detachment may occur.)

F、Adding HRP-Labeled Secondary Antibody Polymer

(1) Shake off excess liquid from tissue sections, add the one-step secondary antibody reagent to completely cover the tissue after the circle drawn by the oil pen is fully visible, and incubate at room temperature in a wet box for 20 minutes;

(2) After secondary antibody incubation, rinse with PBS buffer for 2~3 times, then transfer to a PBS jar and soak for 2 minutes × 3 times.

G、Adding DAB Chromogenic Reagent

(1) Shake off excess liquid from tissue sections, add freshly prepared DAB chromogenic reagent to completely cover the tissue, and incubate at room temperature in a wet box for 5 minutes;

(2) After DAB incubation, shake off the DAB on the slides onto absorbent paper, then put them into distilled water and rinse thoroughly for 3 minutes × 2 times to terminate the staining.

H、Counterstaining and Bluing

(1) After thorough rinsing with distilled water, immerse in hematoxylin staining solution for counterstaining for 8~10 seconds, and rinse clean with distilled water after counterstaining;

(2) After counterstaining, immerse in 1% lithium carbonate solution for bluing for 8~15 seconds, and rinse clean with distilled water after bluing.

(Note: Adjust the counterstaining time according to the intensity of different hematoxylin staining solutions, so that the cell nucleus shows light blue to dark blue. Over-staining or insufficient staining may affect the judgment of results.)

I、Dehydration, Clearing and Mounting

(1) Shake off excess liquid from tissue sections, immerse the tissue sections in 85% ethanol, 95% ethanol and anhydrous ethanol successively for 3 minutes each;

(2) After dehydration, immerse the tissue sections in fresh xylene for 5~10 minutes;

(3) When the tissue sections become transparent, they can be mounted with neutral balsam and coverslips.

5） Result Interpretation:

Immunohistochemical staining results should be observed and interpreted by experienced professionals under an optical microscope for the stained sections.

【Quality Control】

Before interpreting the staining results of the tested specimens, the staining results of a series of quality controls such as reagent control and tissue control should be analyzed and confirmed, compared with the known positive and negative results, and referred to the previous quality control staining results. Only when a series of controls show staining results consistent with the known ones, the staining results of the tested specimens are considered valid. A complete immunohistochemical experiment should set up positive tissue control, negative tissue control and blank (or negative) reagent control to ensure that the entire experimental process is accurate and controllable.

Positive Tissue Control should be fresh biopsy or surgical specimens, fixed, pretreated and made into paraffin-embedded sections by the same method as the tested specimens. The ideal positive tissue control should be known to contain both high and low expression of the tested antigen to ensure that the experimental results can reflect different antigen expression levels in different tested specimens, especially to avoid missing cases with low expression. Normal staining results of positive tissue control indicate that the tissue preparation of the tested specimen is correct and the staining method is appropriate. A positive tissue control should be set up for each staining experiment. If the positive tissue control fails to show the correct positive staining, the staining results of the tested specimens in this experiment should be considered invalid. (Note: Normal staining results of positive tissue control only indicate that the experimental process and reagent performance meet the requirements, and cannot be used as an evaluation standard for the specificity of the staining results of the tested specimens.)

Negative Tissue Control should also be fixed, pretreated and made into paraffin-embedded sections by the same method as the tested specimens, and is known to have no expression of the tested antigen. A negative tissue control should be set up for each staining experiment to confirm the performance of the antibody and provide a background for specific staining. Most of the time, the tissue sections of the tested specimens contain different cell types, and those negative cells can be used as internal negative tissue controls. If the negative tissue control shows specific positive staining, the staining results of the tested specimens in this experiment should be considered invalid.

Blank (or Negative) Reagent Control can be selected to use PBS buffer or IgG homologous to the primary antibody to replace the primary antibody for the experiment, aiming to evaluate the specific staining of the primary antibody, judge the presence of non-specific staining, and provide a better explanation for the specific staining at the antigen site. The incubation time and temperature of the blank (or negative) control reagent should be consistent with those of the primary antibody.

【Interpretation of Staining Results】

Immunohistochemical staining results should be observed and interpreted by experienced professionals under an optical microscope for the stained sections.

A、 Immunohistochemical staining results should be based on the normal results of a series of control stainings, otherwise the staining results of this experiment should be considered invalid.

B、 Positive Staining Result (+): On the basis of normal results of a series of control stainings, brown-yellow staining is seen at the specific site of specific cells in the tested tissue sections without background staining, indicating the presence and expression of the tested antigen.

C、 Negative Staining Result (-): On the basis of normal results of a series of control stainings, no brown-yellow staining is seen in specific cells in the tested tissue sections, indicating no expression or extremely low expression of the tested antigen.

【Limitations of Staining Method】

1） Changes in specimen fixation and pretreatment, tissue antigen retrieval, antibody incubation temperature and time or other experimental conditions may affect the correctness of staining results.

2） In each staining process, blank (or negative) reagent control, positive tissue control and negative tissue control should be set up, and all control staining results should be normal, otherwise the experiment is considered invalid.

3） A complete immunohistochemical experiment process includes multiple steps, including reagent selection, tissue selection, fixation and treatment, section preparation, staining and result interpretation. The treatment method of tissues before staining can directly affect the staining effect. Improper fixation, freezing, thawing, cleaning, drying, sectioning or contamination by other tissues or liquids may cause false positive, inaccurate antibody localization or false negative results. Different fixation and embedding methods or irregularities within the tissue may also cause abnormal staining results. At the same time, excessive or insufficient counterstaining may affect the correct interpretation of results.

4） Interpretation of immunohistochemical staining results should be combined with pathological morphology and other data. Any interpretation of staining or its absence should be supplemented by morphology, correct controls and other tests.

5） The reagent may have unexpected reactions in untested tissues. Due to the biological variability of antigen expression in tumors or other pathological tissues, it is impossible to completely eliminate the possibility of unexpected reactions in the tested tissues.

6） False positive results may be caused by non-immunological binding of protein or substrate reaction products. They may also be caused by red blood cells and cytochrome C.

【Precautions】

1） This product is for research use only, not for other purposes.

2） This product should be used by professionals.

3） This product contains materials of biological origin, and its treatment should comply with relevant requirements.

4） Take appropriate protective measures to avoid contact of the reagent with skin and eyes.

5） Whether this product is suitable for non-formalin fixed tissues has not been confirmed.

6） This product contains sodium azide as a preservative. Sodium azide can react with lead and copper to form explosive metal azides. To prevent the formation of metal azides, rinse with a large amount of water to avoid their accumulation.

【References】

[1] Moll R, et al. Differentiation, 1993, 53(2):75-93.

[2] Calnek D, et al. Differentiation, 1993, 53(2):95-104.

[3] Su YC, et al. Kaohsiung J Med Sci, 2006, 22(1):14-19.

【Manufacturer】

Guangzhou Myabtech Biological Inc.

Myabtech Biological Inc.