Universal Enhanced IHC Secondary Antibody (HRP-labeled) Product Manual

Universal Enhanced IHC Secondary Antibody (HRP-labeled)

Datasheet

Datasheet No.: MYE02253

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

[Product Name]

[Product No. and Pack Size]

[Intended Use]

This antibody reagent is intended for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections, for use in conjunction with mouse and rabbit primary antibodies.

[Main Components]

Secondary Antibody Solution A

Secondary Antibody Solution B

[Storage Conditions and Shelf Life]

Store the reagent at 2–8°C (not below 0°C), protected from light. Return it to the 2–8°C refrigerator immediately after each use. See the product outer packaging for the shelf life (Note: if the reagent is not stored under the above conditions, its performance may be affected).

[Principle of immunohistochemical staining]

In immunology, antigen and antibody molecules bind specifically due to structural complementarity and mutual affinity. Based on this principle, antibodies bind to specific antigens/proteins in the human tissue and cells being tested. Subsequently, a redox chemical reaction causes the enzyme chromogen of the labeled antibody to develop color, thereby revealing the distribution of antigens within tissues and cells.

First, the antibody specifically binds to the antigen on the tissue to be tested; second, the enzyme-labeled anti-mouse/rabbit IgG polymer recognizes the antibody already bound to the antigen; third, the chromogenic substrate is added, and the horseradish peroxidase on the polymer catalyzes the decomposition of H₂O₂ in the DAB chromogenic solution, oxidizing benzidine to benzidine imine, thereby producing yellow or brown-yellow coloration at the antigen sites in the tissue section; finally, the sample is counterstained and mounted. By observing the color development under a microscope, the location and expression of the antigen in the tissue section can be inferred.

[Sample Requirements]

Fresh biopsy or surgical tissue samples, fixed in 10% neutral buffered formalin for 6–24 hours, then grossed, dehydrated, and paraffin-embedded into blocks according to technical specifications. Paraffin blocks are stable for 5 years at room temperature.

Section the tissue blocks at 3–5 μm thickness, mount on adhesion-treated slides, and bake in a 58–60°C incubator for 1 hour before staining. Store unused unstained sections at room temperature (15–25°C) (preferably with antioxidant protection). To ensure good reproducibility of antigen distribution in tissue sections, complete IHC staining within 7 days.

[Staining Method]

The following is the recommended configuration and procedure for manual immunohistochemistry. If staining is performed on an automated IHC instrument, adjustments should be made according to the specific instrument and chromogenic detection kit. A complete IHC experiment should include a positive tissue control, a negative tissue control, and a blank (or negative) reagent control. For evaluation and validation of the experimental results, please refer to [Quality Control] and [Interpretation of Staining Results].

1) Required instruments and equipment:

Induction cooker, stainless steel pot, timer, humidified incubation box, staining rack, staining jar, coverslips, light microscope (40×-200×), wash bottle, paper towels, pipettes, etc.

2) Reagent and material preparation:

This antibody reagent working solution requires no dilution before use. For the preparation of PBS buffer, antigen retrieval solution, primary antibody, DAB chromogen solution, and other reagents, please refer to the respective product datasheets.

Other materials required for the experiment are as follows:

PBS buffer (pH 7.2-7.4), antigen retrieval solution, endogenous peroxidase blocking reagent, primary antibody, DAB chromogen 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-coated slides, coverslips, etc.

3) Experimental temperature conditions: room temperature 18°C-28°C.

4) Experimental procedure:

A. Deparaffinization and hydration

(1) Place the tissue sections sequentially in 2 jars of fresh xylene, soaking for 10 minutes each;

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

(3) Rinse gently with distilled water for 1 minute, then soak in distilled water until ready for retrieval.

B. Antigen retrieval: please refer to the relevant primary antibody datasheet for details;

C. Blocking of endogenous peroxidase

(1) Shake off excess liquid from the tissue section and quickly immerse the slide in peroxidase blocker (3% H₂O₂) for 10 minutes;

(2) After blocking, rinse gently with distilled water for 3 minutes × 2 times. After rinsing, soak in distilled water and prepare to draw the circle.

D. Draw the circle

Shake off or carefully wipe away excess liquid around the tissue section. Use an IHC hydrophobic pen to draw a circle around the tissue; the circle should be 2–3 mm from the tissue edge. Ensure the entire circle encloses all tissue, then rinse gently with distilled water for 3 minutes × 2 times. After rinsing off excess pen liquid, soak in PBS buffer and prepare for primary antibody incubation.

E. Apply primary antibody or control reagent

(1) Shake off excess liquid from the tissue section. Once the hydrophobic pen circle is fully visible, apply the primary antibody working solution so that it completely covers the tissue, and incubate in a humid chamber at room temperature. For the specific incubation time, please refer to the relevant primary antibody datasheet.

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

(Note: During the experiment, when washing tissue sections with PBS buffer, do not rinse the tissue surface directly, as this may cause the section to detach.)

F. Add secondary antibody solution A and secondary antibody solution B

(1) Shake off excess liquid from the tissue section. Once the circle drawn with the PAP pen is fully visible, add secondary antibody solution A to completely cover the tissue, and incubate in a humid chamber at 37°C for 10 minutes;

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

(3) Shake off excess liquid from the tissue section. Once the circle drawn with the PAP pen is fully visible, add secondary antibody solution B to completely cover the tissue, and incubate in a humid chamber at 37°C for 10 minutes;

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

G. Add DAB chromogen

(1) Shake off excess liquid from the tissue section, add freshly prepared DAB chromogen to completely cover the tissue, and incubate in a humid chamber at room temperature for 5 minutes;

(2) After DAB incubation, shake the DAB off the slide onto a paper towel, then rinse thoroughly in distilled water for 3 minutes × 2 times to stop the staining.

H. Counterstaining and bluing

(1) After thorough rinsing with distilled water, counterstain in hematoxylin staining solution for 8–10 seconds, then rinse clean with distilled water;

(2) After counterstaining, place in 1% lithium carbonate solution for 8–15 seconds to blue, then rinse clean with distilled water.

(Note: Adjust the counterstaining time according to the strength of different hematoxylin staining solutions. The nuclei should appear light blue to dark blue; overstaining or understaining may affect interpretation of the results.)

I. Dehydration, clearing, and mounting

(1) Shake off excess liquid from the tissue section, and sequentially immerse the tissue section in 85% ethanol, 95% ethanol, and absolute ethanol for 3 minutes each;

(2) After dehydration, place the tissue section in fresh xylene and soak for 5–10 minutes;

(3) Once the tissue section appears translucent, mount with neutral balsam and a coverslip.

5) Interpretation of results:

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

[Quality Control]

Before interpreting the staining results of the test specimen, the staining results of a series of controls, such as reagent controls and tissue controls, should first be analyzed and confirmed by comparing them with known positive and negative results and referring to previous quality control staining results. Only when all controls show staining results consistent with the known results can the staining results of the test specimen be considered valid. A complete immunohistochemistry experiment should include a positive tissue control, a negative tissue control, and a blank (or negative) reagent control to ensure that the entire experimental process is accurate and controllable.

Positive tissue control should be a fresh biopsy or surgical specimen fixed, processed, and paraffin-embedded in the same manner as the specimen to be tested. An ideal positive tissue control should be known to contain both high expression and low expression of the antigen under test, so as to ensure that the test results reflect the different antigen expression levels in different specimens to be tested, and in particular to avoid missing cases with low expression. A normal positive tissue control staining result indicates that the tissue preparation of the specimen to be tested is correct and the staining method is appropriate. A positive tissue control must be set up in every staining run. If the positive tissue control fails to show the correct positive staining, the staining results of the specimens to be tested in that run should be considered invalid. (Note: A normal positive tissue control staining result only indicates that the experimental procedure and the performance of the reagents meet the requirements, and cannot serve as a criterion for evaluating the specificity of the staining results of the specimens to be tested.)

Negative tissue control should also be fixed, processed, and paraffin-embedded in the same manner as the specimen to be tested, and should be known not to express the antigen under test. A negative tissue control must be set up in every staining run to confirm the performance of the antibody and to provide a background for specific staining. Most of the time, the tissue section of the specimen to be tested will contain different cell types, and those negative cells can serve as an internal negative tissue control. If specific positive staining appears in the negative tissue control, the staining results of the specimens to be tested in that run should be considered invalid.

Blank (or negative) reagent control: PBS buffer or IgG from the same source as the primary antibody may be used in place of the primary antibody, in order to evaluate the specific staining of the primary antibody, determine whether non-specific staining is present, and provide a better interpretation of specific staining at the antigen site. The incubation time and temperature of the blank (or negative) control reagent should be the same as those of the primary antibody.

[Interpretation of Staining Results]

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

A. IHC staining results should be based on a series of control staining results all being normal; otherwise, the staining results should be considered invalid.

B. Positive staining result (+): On the basis that a series of control staining results are all normal, brown-yellow staining is seen in specific sites of specific cells in the tested tissue section, with no background staining, indicating expression of the tested antigen.

C. Negative staining result (-): On the basis that a series of control staining results are all normal, no brown-yellow staining is seen in specific cells in the tested tissue section, 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 correctness of staining results.

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

3) A complete IHC experiment involves multiple steps, including reagent selection, tissue selection, fixation and processing, section preparation, staining, and interpretation of results. The handling of tissue before staining can directly affect staining quality. Improper fixation, freezing, thawing, washing, drying, sectioning, or contamination by other tissues or fluids may cause false-positive results, inaccurate antibody localization, or false-negative results. Differences in fixation and embedding methods or irregularities within the tissue may also cause abnormal staining results. In addition, excessive or insufficient counterstaining may also affect the correct interpretation of results.

4) Interpretation of IHC 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) Reagents may show unexpected reactions in untested tissues. Because antigen expression in tumors or other pathological tissues is biologically variable, the possibility of unexpected reactions in the tested tissue cannot be completely eliminated.

6) False-positive results may be caused by non-immunological binding of proteins 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) Whether this product is suitable for non-formalin-fixed tissues has not been established.

[References]

1 Wu Bingquan, Liu Yanfang. Immunohistochemical Pathological Diagnosis [M]. Beijing: Beijing Science and Technology Press, 2007.3.

2 Dabbs David J. Diagnostic Immunohistochemistry [M]. Beijing: Peking University Medical Press, 2008.9.

3 Ed Harlow, David Lane. Antibody Technology Laboratory Guide [M]. Translated by Shen Guanxin, Gong Feili, et al. Beijing: Science Press,

2002: 79-80, 105.

[Manufacturer]

Myabtech Biological Inc.