Ki-67 Product Manual

Ki-67 Instructions

Instruction Manual No.:MyM1-Ki67253

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

【Product Name】

【Product Code and Packaging Specifications】

【Intended Use】

This antibody reagent is suitable for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections to identify the expression of Ki-67 protein in tissues.

Ki-67 antigen is a nuclear antigen specifically associated with cell proliferation, mainly used for studying cell proliferation activity. Ki-67 is expressed in proliferating cells during all active phases of the cell cycle (late G1, S, G2, and M phases) but not in quiescent cells. In the G1 phase, it is predominantly localized in the perinucleolar region, and later appears in the nuclear matrix. Ki-67 is thought to be involved in maintaining cell proliferation; however, the mechanism by which it exerts its function remains unclear. Data indicate that the Ki-67 proliferation index is closely related to tumor differentiation, invasion, metastasis, and prognosis. Therefore, it serves as an important reference in tumor research.

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

【Main Components】

Monoclonal mouse anti-human Ki-67 protein antibody, Clone No.: MyM1-Ki67, 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 below 0℃). Return to the 2-8℃ refrigerator immediately after each use. The validity period is indicated on the outer packaging of the product. (Note:Improper storage as specified above may affect the performance of the reagent.)

【Principle of Immunohistochemical Staining】

In immunology, antigen and antibody molecules specifically bind due to structural complementarity and affinity. Based on this principle, the antibody will bind to specific antigens/proteins in the tested human tissues and cells. Subsequently, the enzyme chromogen labeled on the antibody is developed through redox chemical reactions, thereby showing the distribution of antigens 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 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 specimens are fixed in 10% neutral buffered formalin for 6-24 hours, and processed into paraffin blocks by sampling, dehydration and paraffin embedding according to technical specifications. Paraffin blocks can be stored at room temperature for a valid period of 5 years.

Section the paraffin blocks into 3-5μm thick slices, adhere them to poly-L-lysine coated slides, and bake the slides in a constant temperature incubator at 58-60℃ for 1 hour for staining. Unused unstained slides can be stored at room temperature (15-25℃) (preferably with anti-oxidation treatment). For good reproducibility of antigen distribution in tissue sections, it is recommended to complete immunohistochemical staining within 7 days.

【Staining Method】

The following are the recommended preparation and procedures for manual immunohistochemistry. If automated immunohistochemical instruments are used for staining, adjustments should be made according to different instruments and chromogenic kits. A complete immunohistochemical experiment should include positive tissue control, negative tissue control and blank (or negative) reagent control. Evaluation and verification of experimental results should 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, coverslips, optical microscope (40×-200×), wash bottle, absorbent paper, pipette, etc.

2) Reagent and Material Preparation:

No dilution is required for the working solution of this antibody reagent before use. If using the concentrate, it is recommended to dilute it into a working solution with PBS buffer or antibody diluent at a ratio of 1:100 to 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 instructions.

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 (e.g., neutral balsam), poly-L-lysine coated slides, coverslips, etc.

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

4) Experimental Procedures:

A. Deparaffinization and Hydration

(1) Immerse the tissue sections in 2 jars of fresh 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) Place the prepared Tris-EDTA antigen retrieval solution (pH9.0) in a pressure cooker, place the deparaffinized and hydrated tissue sections on a heat-resistant plastic slide rack, and immerse them in the retrieval solution to ensure the tissue sections are completely submerged;

(2) Cover the pressure cooker and 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 switch to medium fire (800W/130℃). After the timing, 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, rinse the cover with running water to cool it down until the automatic lock core falls into place, turn off the running water, remove the pressure limiting valve, open the cover carefully (beware of steam scald), and slowly inject running water into the pot (avoid direct flushing of the tissue sections with running water);

(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 the sections), rinse gently with distilled water 2 times for 3 minutes each, 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 from drying when "shaking off excess liquid from the tissue sections", otherwise the staining effect will be affected.)

C. Blocking of Endogenous Peroxidase

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

(2) After blocking, rinse gently with distilled water 2 times for 3 minutes each, 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 hydrophobic pen at a distance of 2~3mm from the tissue edge. Ensure the entire circle encloses the whole tissue, then rinse gently with distilled water 2 times for 3 minutes each to wash off excess hydrophobic pen solution, and soak in PBS buffer for primary antibody incubation.

E. Addition of Primary Antibody or Control Reagent

(1) Shake off excess liquid from the tissue sections, add the primary antibody working solution to completely cover the tissue after the circle drawn by the hydrophobic 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 2~3 times, then transfer to a PBS jar and soak for 3 times for 2 minutes each.

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

F. Addition of HRP-Labeled Secondary Antibody Polymer

(1) Shake off excess liquid from the tissue sections, add the one-step secondary antibody reagent to completely cover the tissue after the circle drawn by the hydrophobic 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 2~3 times, then transfer to a PBS jar and soak for 3 times for 2 minutes each.

G. Addition of DAB Chromogen

(1) Shake off excess liquid from the tissue sections, add freshly prepared DAB chromogen 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 immerse in distilled water and rinse thoroughly 2 times for 3 minutes each 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 thoroughly with distilled water after counterstaining;

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

(Note: Adjust the counterstaining time according to the intensity of different hematoxylin staining solutions, with the nucleus showing light to dark blue as appropriate. Over-staining or under-staining may affect the interpretation of results.)

I. Dehydration, Clearing and Mounting

(1) Shake off excess liquid from the 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) Mount the tissue sections with neutral balsam and coverslips when they become transparent.

5) Result Interpretation:

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

【Quality Control】

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

Positive Tissue Control should be fresh biopsy or surgical specimens fixed, pretreated and processed into paraffin sections by the same method as the tested specimens. An ideal positive tissue control should be known to contain both high expression 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 the 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 the positive tissue control only indicate that the experimental procedure 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 processed into paraffin sections by the same method as the tested specimens, and 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. In most cases, the tissue sections of the tested specimens contain different cell types, and those negative cells can serve 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 performed by replacing the primary antibody with PBS buffer or IgG homologous to the primary antibody, aiming to evaluate the specific staining of the primary antibody, determine 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 under an optical microscope by experienced professionals.

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 observed at the specific site of specific cells in the tested tissue sections without background staining, indicating the presence 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 observed in specific cells of the tested tissue sections, indicating no expression or extremely low expression of the tested antigen.

【Limitations of the Staining Method】

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

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

3) A complete immunohistochemical experiment process includes multiple steps, including reagent selection, tissue selection, fixation and processing, section preparation, staining and result interpretation. The processing method of tissues before staining can directly affect the staining effect. Improper fixation, freezing, thawing, washing, 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, over-counterstaining 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, proper controls and other tests.

5) The reagent may produce unintended reactions in untested tissues. Due to the biological variability of antigen expression in tumors or other pathological tissues, the possibility of unintended reactions in the tested tissues cannot be completely eliminated.

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 and not for other purposes.

2) This product must be used by professionals.

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

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

5) The suitability of this product for non-formalin fixed tissues has not been confirmed.

6) This product contains sodium azide as a preservative, which 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 accumulation.

【References】

1.Mckeever P, et al., J Neuropathol Exp Neurol 1998; 57:931-936.

2.Coons SW, et al., Neurosurgery 1997 Oct; 41(4):878-84.

3.Allegra CJ, et al., J Clin Oncol. 2003 Jan 15; 21(2):241-50.

【Manufacturer】

Guangzhou Myabtech Biological Technology Co., Ltd.

Myabtech Biological Inc.