Goat Anti-Rabbit IHC Secondary Antibody Reagent (AP-labeled) Product Manual

Goat anti-rabbit IHC secondary antibody reagent (AP-labeled)

Datasheet

Datasheet No.: MYE05253

(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 use in conjunction with rabbit-derived primary antibodies.

[Main components]

·[Storage conditions and shelf life]

Store the reagent at 2-8°C (not below 0°C), protected from light. After each use, return it immediately to the 2-8°C refrigerator. 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 IHC Staining]

In immunology, antigens and antibodies can bind specifically due to structural complementarity and mutual affinity. Based on this principle, antibodies will bind to specific antigens/proteins in the human tissue or cells to be tested. Subsequently, a redox reaction causes the enzyme-labeled antibody's chromogen to develop color, revealing the distribution of antigens in tissues and cells.

First, the rabbit-derived primary antibody specifically binds to the antigen on the tissue to be tested; second, the alkaline phosphatase (AP)-labeled goat anti-rabbit IgG polymer recognizes and binds to the primary antibody already attached to the antigen; third, the chromogenic substrate is added, and the AP on the polymer catalyzes the decomposition of naphthol AS-MX phosphate in the Fast-red chromogen solution to produce naphthol, which then binds with Fast-red TR to form an insoluble red precipitate, producing red 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 formalin solution 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 specimen blocks at 3–5 μm thickness, adhere to adhesion-treated slides, and bake in a 58–60°C incubator for 1 hour in preparation for staining. Store unused sections at room temperature (15–25°C) (preferably with antioxidant protection); to ensure good reproduction of antigen distribution in tissue sections, complete IHC staining within 7 days.

[Staining Procedure]

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 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, 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 preparation of PBS buffer, antigen retrieval solution, primary antibody, Fast-red 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), Tris-EDTA antigen retrieval solution (0.01 M, pH 9.0), endogenous peroxidase blocking reagent, primary antibody, Fast-red chromogen, 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, then soak in distilled water before drawing the circle.

D. Draw the circle

Shake off or carefully wipe away excess liquid around the tissue section, and draw a circle around the tissue with an IHC pen at a distance of 2–3 mm from the tissue edge. Ensure the entire circle encloses all tissue, then rinse gently with distilled water for 3 minutes × 2. After rinsing off excess oil, soak in PBS buffer before primary antibody incubation.

E. Apply primary antibody or control reagent

(1) Shake off excess liquid from the tissue section. Once the 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 incubation time, refer to the instructions of the relevant primary antibody reagent.

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

(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 AP-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 AP-labeled 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 Fast-red chromogen

(1) Shake off excess liquid from the tissue section, add freshly prepared Fast-red chromogen so that it completely covers the tissue, and incubate in a humid chamber at room temperature in the dark for 15 minutes;

(2) After Fast-red incubation, shake the Fast-red 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:

IHC 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, including reagent controls and tissue controls, should first be analyzed and confirmed by comparison with known positive and negative results and with reference 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 IHC 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 controlled.

The 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, so as 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 of the specimen to be tested was properly prepared and the staining method was 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, and cannot serve as a criterion for evaluating the specificity of the staining results of the specimens to be tested.)

The negative tissue control should also be fixed, processed, and paraffin-embedded in the same manner as the specimen to be tested, and is known not to 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 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.

For the blank (or negative) reagent control, PBS buffer or IgG of the same origin as the primary antibody may be used instead 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 the 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]

The interpretation of immunohistochemical staining results should be performed by experienced professionals who examine and evaluate the stained sections under a light microscope.

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

B. Positive staining result (+): On the basis of a series of control staining results all being normal, red staining is observed at specific sites of specific cells in the tested tissue section, with no background staining, indicating that the tested antigen is expressed.

C. Negative staining result (-): On the basis of a series of control staining results all being normal, no red staining is observed in specific cells in the tested tissue section, indicating that the tested antigen is not expressed or is expressed at an extremely low level.

[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 the 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 immunohistochemical experiment involves multiple steps, including reagent selection, tissue selection, fixation and processing, section preparation, staining, and interpretation of results. The manner in which tissue is processed before staining can directly affect the staining outcome. 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) The interpretation of immunohistochemical staining results should be made in conjunction with pathological morphology and other data. The interpretation of any staining or its absence should be supplemented by morphology, proper controls, and other tests.

5) The reagent 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 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.

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 Zhao Xiaoming, Li Guangqian, Liu Xiaoliu, et al. Immunohistochemical study of the coexistence of androgen receptor and choline acetylase in rat intracardiac ganglion cells [C]// Chinese Society for Anatomical Sciences. Acta Anatomica Sinica — Abstracts of the 2002 Annual Meeting of the Chinese Society for Anatomical Sciences. Acta Anatomica Sinica Press, 2002:155.

[Manufacturer]

Myabtech Biological Inc.