P16 Product Manual

P16 Manual

Manual No.:MyM1-p16253

(For research use only, not for diagnostic procedures)

【Product Name】

【Product No. 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 P16 protein in tissues.

P16, also known as "Mitotic Inhibitor", is one of the cell cycle regulatory proteins. Its function is to competitively bind CDK4/6 with cyclin D, preventing cells from progressing from the G1 phase to the S phase, thereby negatively regulating mitosis. In tumor cells, due to gene mutation or deletion, P16 expression often increases. P16 is closely related to HPV infection-induced cervical cancer. The P16 staining result in High-grade Squamous Intraepithelial Lesions (HSIL) is often strong and complete in the cervical epithelium, while weak to moderate staining tends to be the result of Low-grade Squamous Intraepithelial Lesions (LSIL). Normal cervical epithelium shows almost no expression. Therefore, P16 is helpful for studying the grading of cervical intraepithelial neoplasia.

(Note: The interpretation of immunohistochemical staining results should be combined with pathological morphology and other data. This staining result is not a diagnostic indicator.)

【Main Components】

Monoclonal mouse anti-human P16 protein antibody, Clone No.: MyM1-p16, 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 (N_aN₃) and protein stabilizers.

【Storage Conditions and Shelf Life】

Store at 2-8°C (do not freeze) and protect from light. Return to 2-8°C refrigerator immediately after each use. The expiration date is indicated on the outer package. (Note: Failure to store the reagent under the specified conditions may affect its performance).

【Principle of Immunohistochemical Staining】

In immunology, antigen and antibody molecules bind specifically due to complementary structures and mutual affinity. Based on this principle, the antibody will bind to specific antigens/proteins in the tested human tissue and cells. Subsequently, the enzyme chromogen linked to the antibody is visualized through a redox reaction, revealing the distribution of antigens within the tissue and cells.

First, this antibody specifically binds to the antigen on the test tissue. Second, the enzyme-labeled anti-mouse/rabbit IgG polymer recognizes the antibody already bound to the antigen. Third, upon adding the chromogenic substrate, the horseradish peroxidase on the polymer catalyzes the decomposition of H₂O₂ in the DAB solution, oxidizing benzidine to benzidine imine, resulting in yellow or brownish-yellow coloration at the antigen sites in the tissue section. Finally, the sample is counterstained and mounted. The staining is observed under a microscope to infer the presence, location, and expression level of the antigen in the tissue section.

【Specimen Requirements】

Fresh biopsy or surgical tissue specimens should be fixed in 10% neutral buffered formalin for 6-24 hours, then processed, dehydrated, and embedded in paraffin blocks according to standard procedures. Paraffin blocks can be stored at room temperature for up to 5 years.

Sections of 3-5μm thickness should be cut from the specimen blocks, mounted on adhesive-coated slides, and baked in a 58-60°C oven for 1 hour before staining. Unstained slides not used immediately can be stored at room temperature (15-25°C) (preferably under oxidation protection). For optimal antigen detection, immunohistochemical staining is recommended within 7 days.

【Staining Procedure】

The following recommendations and steps are for manual IHC staining. If using an automated IHC stainer, adjustments should be made according to the specific instrument and detection kit used. A complete IHC run should include positive tissue control, negative tissue control, and blank (or negative) reagent control. For evaluation and validation of experimental results, refer to 【Quality Control】 and 【Interpretation of Staining Results】.

1) Required Instruments and Equipment:

Induction cooker, stainless steel pressure cooker, timer, humidified chamber, staining rack, staining jars, coverslips, light microscope (40×-200×), wash bottle, absorbent paper, pipettes, etc.

2) Reagents and Materials Preparation:

The ready-to-use antibody reagent does not require dilution before use. If using the concentrate, it is recommended to dilute it with PBS buffer or antibody diluent to a working concentration of 1:100-1:200. Please refer to the respective product manuals for the preparation of PBS buffer, antigen retrieval solution, secondary antibody, and DAB substrate solution.

Other materials required for the experiment are as follows:

PBS buffer (pH 7.2-7.4), Tris-EDTA Antigen Retrieval Solution (pH 9.0), Endogenous Peroxidase Blocking Agent, HRP-conjugated anti-mouse/rabbit IgG polymer, DAB substrate solution, Hematoxylin staining solution, blank (or negative) control reagent, positive and negative control tissue sections, xylene or xylene substitute, ethanol (absolute, 95%, 85%), distilled or deionized water, mounting medium (e.g., neutral balsam), adhesive slides, coverslips, etc.

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

4) Experimental Steps:

A.Deparaffinization and Rehydration

(1) Place tissue sections sequentially in 2 changes of fresh xylene, incubate for 10 minutes each;

(2) Place tissue sections sequentially in absolute ethanol, 95% ethanol, 85% ethanol, incubate for 5 minutes each;

(3) Rinse gently with distilled water for 1 minute, then immerse in distilled water for antigen retrieval.

B.Antigen Retrieval (Heat-induced using pressure cooker)

(1) Place prepared Tris-EDTA Antigen Retrieval Solution (pH 9.0) into a pressure cooker. Place the deparaffinized and rehydrated tissue sections on a heat-resistant plastic slide rack and immerse them in the retrieval solution, ensuring sections are completely covered;

(2) Close the lid securely, set the pressure valve, and heat on high (1600W/210°C) until steam escapes. Start timing for 2 minutes from the moment steam escapes, then reduce heat to medium (800W/130°C). After timing ends, turn off the heat source and carefully move the pressure cooker away from the hot plate. Allow it to cool naturally for 5 minutes;

(3) Carefully transfer the pressure cooker to a sink. Run cold tap water over the lid, allowing it to cool until the safety lock drops. Turn off the water, remove the pressure valve, and carefully open the lid (beware of steam). Slowly add cold water to the cooker (avoid directing the stream onto the slides);

(4) When the temperature drops below 40°C (water feels warm but not hot to the touch), remove the slide rack and immediately immerse in distilled water (to prevent drying). Rinse gently with distilled water for 3 minutes ×2 times, then immerse in distilled water for blocking.

(Note: Tissue sections are prone to drying after antigen retrieval. In subsequent steps involving "shaking off excess liquid", be careful not to let the tissue dry, as this may affect staining results.)

C.Blocking Endogenous Peroxidase

(1) Shake off excess liquid from slides and immediately immerse them in Endogenous Peroxidase Blocking Agent (3% H₂O₂) for 10 minutes;

(2) After blocking, rinse gently with distilled water for 3 minutes ×2 times, then immerse in distilled water for barrier drawing.

D.Barrier Drawing

Shake off or carefully wipe excess liquid from around the tissue section. Use an IHC PAP pen to draw a hydrophobic barrier around the tissue, approximately 2~3mm from the tissue edge. Ensure the entire tissue is encircled, then rinse gently with distilled water for 3 minutes ×2 times to remove excess ink. Immerse slides in PBS buffer for primary antibody incubation.

E.Primary Antibody or Control Reagent Application

(1) Shake off excess liquid from slides. Once the PAP pen circle is clearly visible, apply enough primary antibody working solution to completely cover the tissue section. Incubate in a humidified chamber at room temperature for 30 minutes;

(2) After primary antibody incubation, rinse with PBS buffer 2~3 times, then immerse in a PBS bath for 2 minutes ×3 washes.

(Note: When rinsing tissue sections with PBS buffer during the procedure, avoid directing the stream onto the tissue surface to prevent detachment.)

F.HRP-conjugated Polymer (Secondary Antibody) Application

(1) Shake off excess liquid from slides. Once the PAP pen circle is clearly visible, apply enough ready-to-use secondary antibody polymer to completely cover the tissue section. Incubate in a humidified chamber at room temperature for 20 minutes;

(2) After secondary antibody incubation, rinse with PBS buffer 2~3 times, then immerse in a PBS bath for 2 minutes ×3 washes.

G.DAB Substrate Application

(1) Shake off excess liquid from slides. Apply enough freshly prepared DAB substrate solution to completely cover the tissue section. Incubate in a humidified chamber at room temperature for 5 minutes;

(2) After DAB incubation, tap slides onto absorbent paper to remove excess DAB, then rinse thoroughly in distilled water for 3 minutes ×2 times to stop the chromogenic reaction.

H.Counterstaining and Bluing

(1) After thorough rinsing with distilled water, immerse slides in Hematoxylin staining solution for 8~10 seconds. Rinse with distilled water until clean;

(2) After counterstaining, immerse in 1% Lithium Carbonate solution for 8~15 seconds for bluing. Rinse with distilled water until clean.

(Note: Adjust counterstaining time according to the strength of the Hematoxylin used. The nuclei should appear light to dark blue. Over- or under-staining may affect interpretation.)

I.Dehydration, Clearing, and Mounting

(1) Shake off excess liquid from slides. Place tissue sections sequentially in 85% ethanol,95% ethanol, and absolute ethanol, incubating for 3 minutes in each;

(2) After dehydration is complete, place tissue sections in fresh xylene for 5~10 minutes;

(3) Once tissue sections appear translucent, mount them using neutral balsam and a coverslip.

5) Interpretation of Results:

Immunohistochemical staining results should be observed and interpreted by experienced professionals under a light microscope.

【Quality Control】

Before interpreting the staining results of test specimens, the staining results of reagent controls and tissue controls should be analyzed and confirmed. Compare with known positive and negative results, and refer to previous quality control staining results. Only when all controls show staining results consistent with known patterns should the staining results of the test specimen be considered valid. A complete IHC run 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, processed, and embedded in paraffin using the same method as the test specimens. An ideal positive tissue control should contain both high and low expression levels of the target antigen to ensure the experimental results can reflect different antigen expression levels in various test specimens, particularly avoiding missing cases with low expression. Normal staining of the positive tissue control indicates that the test tissue specimens were prepared correctly and the staining method was appropriate. A positive tissue control must be included in each staining run. If the positive tissue control fails to show correct positive staining, the staining results for the test specimens in that run should be considered invalid. (Note: Normal staining of the positive tissue control only confirms that the experimental procedure and reagent performance meet requirements; it does not serve as a criterion for evaluating the specificity of the test specimen staining.)

Negative Tissue Control should also be fixed, processed, and embedded in paraffin using the same method as the test specimens and should be known to lack expression of the target antigen. A negative tissue control must be included in each staining run to confirm antibody specificity and to provide a baseline for specific staining background. Often, the test tissue section itself may contain different cell types; those negative cells can serve as internal negative controls. If the negative tissue control shows specific positive staining, the staining results for the test specimens in that run should be considered invalid.

Blank (or negative) reagent control is performed by substituting the primary antibody with PBS buffer or an irrelevant IgG of the same isotype. The purpose is to assess non-specific staining and to provide a better interpretation of specific antigen site staining. The incubation time and temperature for the blank (or negative) control reagent should be identical to those for the primary antibody.

【Interpretation of Staining Results】

Immunohistochemical staining results should be observed and interpreted by experienced professionals under a light microscope.

A. Immunohistochemical staining results should be interpreted based on the normal staining of a series of controls. Otherwise, the staining results for that run should be considered invalid.

B. Positive Staining Result (+): Based on normal staining of all controls, brownish-yellow coloration is observed in the specific cellular location of the test tissue section, with no background staining, indicating expression of the target antigen.

C. Negative Staining Result (-): Based on normal staining of all controls, no brownish-yellow coloration is observed in the specific cells of the test tissue section, indicating absence or very low expression of the target antigen.

【Limitations of the Staining Procedure】

1) Specimen fixation and pre-treatment, antigen retrieval method, antibody incubation temperature and time, or changes in other experimental conditions may affect the accuracy of staining results.

2) In each staining run, a blank (or negative) reagent control, positive tissue control, and negative tissue control should be included. All control staining results must be normal; otherwise, the run is considered invalid.

3) The complete IHC procedure involves multiple steps, including reagent selection, tissue selection, fixation and processing, slide preparation, staining, and result interpretation. Tissue handling before staining directly impacts the staining outcome. Improper fixation, freezing, thawing, washing, drying, sectioning, or contamination by other tissues or fluids can lead to false positives, inaccurate antibody localization, or false negatives. Variations in fixation and embedding methods or irregularities within the tissue itself may also cause aberrant staining results. Additionally, over- or under-counterstaining can affect the correct interpretation of results.

4) Interpretation of IHC staining results must be correlated with histomorphology and other clinical data. Any interpretation of staining or lack thereof should be supplemented by morphological analysis, appropriate controls, and other tests.

5) The reagent may show unexpected reactions in untested tissues. Due to the biological variability of antigen expression in tumors or other pathological tissues, the possibility of unexpected reactions in the tested tissue cannot be completely ruled out.

6) False positive results may arise from non-immunological binding of proteins or substrate reaction products. They may also be caused by endogenous substances such as red blood cells and cytochrome C.

【Precautions】

1) For research use only. Not for any other use.

2) This product should be used by trained professionals only.

3) This product contains materials of biological origin. Handling should comply with relevant regulations.

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 established.

6) This product contains sodium azide as a preservative. Sodium azide can react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, flush with large volumes of water to prevent the accumulation of azide compounds.

【References】

[1]  Steiner M S, et al. Oncogene, 2000, 19: 1297-306.

[2]  Guan KL, et. al. Genes and Development, 1994, 8(24): 2939-52.

【Manufacturer】

Guangzhou Myabtech Biological Co., Ltd.

Myabtech Biological Inc.