Universal IHC Secondary Antibody (HRP-labeled)

Universal IHC Secondary Antibody (HRP-labeled)

Datasheet

Datasheet No.: MYE01253

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

[Product Name]

[Product No. and Packaging Specifications]

[Intended Use]

This immunohistochemistry detection system is intended for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections and is used in combination with mouse and rabbit primary antibodies.

[Main Components]

[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 chromogen of the enzyme-labeled antibody to develop color, thereby revealing the distribution of the antigen within the tissue and cells.

First, the primary antibody specifically binds to the antigen on the tissue to be tested; second, the HRP-labeled secondary antibody polymer recognizes the antibody already bound to the antigen; third, chromogenic substrate is added, and the horseradish peroxidase on the polymer catalyzes the decomposition of H₂O₂ in the DAB chromogen solution, oxidizing benzidine to benzidine imine, thereby producing yellow or brown-yellow coloration at 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 when stored 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) (antioxidant treatment recommended). To ensure good reproducibility of antigen distribution in tissue sections, complete IHC staining within 7 days.

[Staining Procedure]

The following are the recommended configuration and steps for manual immunohistochemistry. If staining is performed on an automated IHC instrument, adjustments should be made according to the specific instrument and 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 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 reagent is a ready-to-use working solution; no dilution is required before use. For the preparation of PBS buffer, antigen retrieval solution, primary antibody, DAB chromogen, and other reagents, please refer to the respective product datasheets.

Other materials required for the experiment are listed below:

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 two 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 ready for retrieval.

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

C. Blocking of endogenous peroxidase

(1) Shake off excess liquid from the tissue section and quickly immerse the slide in peroxidase blocking reagent (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 a circle.

D. Draw a 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, positioning the circle 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 oil, soak in PBS buffer and prepare for primary antibody incubation.

E. Add primary antibody or control reagent

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

(2) After primary antibody incubation, rinse with PBS buffer 2–3 times, then transfer to a PBS tank 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 HRP enzyme-labeled secondary antibody polymer

(1) Shake off excess liquid from the tissue section; once the circle drawn with the PAP pen is fully visible, add the one-step secondary antibody reagent so that it completely covers the tissue, and incubate in a humid chamber at room temperature for 20 minutes;

(2) After secondary antibody incubation, 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 so that it completely covers 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, place in hematoxylin staining solution for counterstaining for 8–10 seconds, then rinse clean with distilled water;

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

(Note: Adjust the counterstaining time according to the strength of different hematoxylin staining solutions; 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, then place the tissue section sequentially in 85% ethanol, 95% ethanol, and absolute ethanol, soaking 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 transparent, mount with neutral balsam and a coverslip.

5) Interpretation of results:

The results of immunohistochemical staining 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, including reagent controls and tissue controls, should first be analyzed and confirmed, compared with known positive and negative results, and referenced against 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 test specimen. An ideal positive tissue control should be known to contain both high expression and low expression of the antigen under test, so that the test results can reflect different antigen expression levels among different test specimens, and in particular can avoid missing low-expression cases. A normal staining result in the positive tissue control indicates that the test specimen tissue was properly prepared and the staining method was appropriate. A positive tissue control must be included in every staining run. If the positive tissue control fails to show the correct positive staining, the staining results of the test specimens in that run should be considered invalid. (Note: A normal staining result in the positive tissue control only indicates that the experimental procedure and reagent performance meet requirements; it cannot serve as a criterion for evaluating the specificity of the staining results of the test specimens.)

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

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

4) Use appropriate protective measures to avoid contact of reagents 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. Using Antibodies: A Laboratory Manual [M]. Translated by Shen Guanxin, Gong Feili, et al. Beijing: Science Press,

2002: 79-80, 105.

[Manufacturer]

Myabtech Biological Inc.