Goat Anti-Mouse IHC Secondary Antibody Reagent (HRP-labeled) Product Manual

Goat Anti-Mouse IHC Secondary Antibody (HRP-labeled)

Datasheet

Datasheet No.: MYE06253

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

[Product Name]

[Product No. and Packaging Specifications]

[Intended Use]

This antibody reagent is intended for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections, for binding mouse-derived primary antibodies.

[Main components]

[Storage conditions and shelf life]

Store the reagent at 2–8°C (do not allow it to drop below 0°C), protected from light. After each use, return it to the 2–8°C refrigerator immediately. 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, antigens and antibodies can bind specifically due to structural complementarity and mutual affinity. Based on this principle, antibodies bind to specific antigens/proteins in the human tissue or cells being tested. Subsequently, a redox reaction causes the enzyme chromogen labeled on the antibody to develop color, thereby revealing the distribution of antigens in tissues and cells.

First, the mouse-derived primary antibody specifically binds to the antigen on the tissue to be tested; next, the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG polymer recognizes and binds to the primary antibody already bound to the antigen; then, a chromogenic substrate is added, and the HRP on the polymer catalyzes the decomposition of H₂O₂ in the DAB chromogenic solution, causing benzidine to be oxidized and to form 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 sampled, dehydrated, and paraffin-embedded into blocks according to technical specifications. Paraffin blocks are valid 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) (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, adjust 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 results, refer to Quality Control and Interpretation of Staining Results.

1) Required instruments and equipment:

Induction cooker, pressure cooker, 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 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 listed below:

PBS buffer (pH 7.2 ~ 7.4), Tris-EDTA antigen retrieval solution (0.01 M, pH 9.0), 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 ~ 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 ready for retrieval.

B. Antigen retrieval: please refer to the relevant primary antibody reagent datasheet 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 the circle.

D. Draw the circle

Shake off or carefully wipe away excess liquid around the tissue section. Use an IHC oil 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 oil 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, 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 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 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, compared with known positive and negative results, and referenced against previous quality control staining results. Only when a series of controls all 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.

A positive tissue control should be a fresh biopsy or surgical specimen that has been fixed, processed, and paraffin-embedded in the same manner as the specimen to be tested. An ideal positive tissue control should contain both known cases of high and low expression of the antigen to be detected, to ensure that the test results can 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 for 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; it cannot serve as a criterion for evaluating the specificity of the staining results of the specimens to be tested.)

A negative tissue control should also be fixed, processed, and paraffin-embedded in the same manner as the specimen to be tested, and is known to not express the antigen to be detected. A negative tissue control must be set up for every staining run to confirm the performance of the antibody and to provide a background for specific staining. Most of the time, the tissue sections of the specimens 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.

A blank (or negative) reagent control can be performed using PBS buffer or IgG from the same source as the primary antibody 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, positive tissue control, and 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, slide preparation, staining, and interpretation of results. The manner of tissue processing before staining can directly affect staining performance. 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. At the same time, 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 their 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. Antibodies: A Laboratory Manual [M]. Translated by Shen Guanxin, Gong Feili, et al. Beijing: Science Press, 2002: 79-80, 105.

4 Ni Canrong, Ma Dalie, Zhu Minghua. New progress in the application of immunohistochemistry techniques in pathological diagnosis [J]. Chinese Journal of Clinical and Experimental Pathology, 2007(03):346-349. DOI:10.13315/j.cnki.cjcep.2007.03.033.

5 Zhu Libin, Li Zhongrong. Selection and comparison of several immunohistochemical chromogenic systems [J]. Journal of Wenzhou Medical College, 2006(02):169-171.

6 Zeng Huiling. Direct rapid immunohistochemical method for detection of rabies virus antigen [D]. Jilin University, 2017.

Manufacturer

Myabtech Biological Inc.