Universal Enhanced IHC Secondary Antibody PLUS (HRP-conjugated) Product Manual

Universal Enhanced IHC Secondary AntibodyPLUS(HRPlabeled)

Instruction Manual

Manual No.:MYE07253

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

[Product Name]

[Product No. and Packaging Specifications]

[Intended Use]

This antibody reagent is suitable for formalin-fixed, paraffin-embedded human tissue sections for immunohistochemical staining,for binding mouse and rabbit primary antibodies.

[Main Components]

Secondary AntibodyASolution

Secondary AntibodyBSolution

[Storage Conditions and Validity Period]

The reagent should be stored in the dark at 2-8℃(not below 0℃)storage.Return to the 2-8℃ refrigerator immediately after each use.The validity period is shown on the outer packaging(Note: If the reagent is not stored under the above conditions,may affect its performance).

[Principle of Immunohistochemical Staining]

In immunology, antigen and antibody molecules specifically bind due to structural complementarity and affinity. Based on this principle, the antibody will bind to specific antigens/proteins in the tested human tissues and cells. Subsequently, a redox reaction is used to develop color with the enzyme chromogen labeled on the antibody, thereby showing antigen distribution in tissues and cells.

Firstly, this antibody specifically binds to the antigen on the tested tissue; secondly, the enzyme-labeled anti-mouse/rabbit IgG polymer recognizes the antibody already bound to the antigen; thirdly, after adding the chromogenic substrate, horseradish peroxidase on the polymer can catalyze H₂O₂ decomposition in the DAB chromogen, oxidizing benzidine into benzidine imine, so that yellow or brown-yellow staining appears at antigen sites in the tissue sections; finally the sample is counterstained and mounted. The presence and expression of the antigen on the tissue section are inferred by observing the color development under a microscope.

[Sample Requirements]

Fresh biopsy or surgical specimens are fixed with 10% neutral formalin solution for 6-24 hours, and processed into wax blocks by sampling, dehydration and paraffin embedding according to technical specifications. Wax blocks are valid for 5 years when stored at room temperature.

Section the specimen wax blocks to a thickness of 3-5μm, adhere them to anti-drop slides, and bake the slides in a constant temperature incubator at 58-60℃ for 1 hour for staining. Unused blank slides should be stored at room temperature (15-25℃) (preferably with anti-oxidation treatment). To well reproduce antigen distribution in tissue sections, immunohistochemical staining is recommended within 7 days.

[Staining Method]

The following are the recommended configuration and procedures for manual immunohistochemistry. If automated immunohistochemical instruments are used for staining, please adjust according to different instruments and chromogenic kits. A complete immunohistochemical experiment should set up a positive tissue control, a negative tissue control and a blank (or negative) reagent control. For evaluation and verification of experimental results, please refer to [Quality Control] and [Interpretation of Staining Results].

1) Required Instruments and Equipment:

Induction cooker, stainless steel pot, timer, incubation wet box, staining rack, staining jar, coverslips, optical microscope (40×-200×), wash bottles, absorbent paper, pipettes, etc.

2) Reagent and Material Preparation:

No dilution is required for the working solution of this antibody reagent before use.For preparation of PBS buffer, antigen retrieval solution, primary antibody, DAB chromogen and other reagents, refer to each product manual.

Other materials required for the experiment are as follows:

PBSBuffer(pH7.2-7.4), Antigen Retrieval Solution, Endogenous Peroxidase Blocker, Primary Antibody, DAB Chromogen, Hematoxylinstaining solution, blank (or negative) control reagent, positive and negative control tissue sections, xylene or xylene substitute, ethanol (anhydrous, 95%, 85%), distilled or deionized water, mounting medium (such as neutral gum), anti-drop slides, coverslips, etc.

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

4) Experimental Procedures:

A, Deparaffinization and Hydration

(1) Immerse the tissue sections in 2 jars of fresh xylene successively for 10 minutes;

(2) Immerse the tissue sections in anhydrous ethanol, 95% ethanol, 85% ethanol successively for 5 minutes;

(3) Rinse gently with distilled water for 1 minute, and soak in distilled water for retrieval after rinsing.

B, Antigen Retrieval:Please refer to the relevant primary antibody reagent Instruction Manual;

C, Blocking of Endogenous Peroxidase

(1) Shake off excess liquid from the tissue sections, place the slides in peroxidase blocker (3% H₂O₂) and soak for 10 minutes;

(2) After blocking, rinse gently with distilled water for 3 minutes ×2 times, and soak in distilled water for circle drawing after rinsing.

D, Circle Drawing

Shake off or carefully wipe off excess liquid around the tissue sections, draw a circle around the tissue with an immunohistochemical oil pen. The circle should be 2~3mm from the tissue edge; after ensuring the circle encloses all tissue, rinse gently with distilled water for 3 minutes ×2 times, and after rinsing off excess oil, soak in PBSBuffer for primary antibody incubation.

E, Adding Primary Antibody or Control Reagent

(1) Shake off excess liquid from the tissue sections, add the primary antibody working solution to completely cover the tissue after the circle drawn by the oil pen is fully visible, and incubate at room temperature in a wet box for ,For incubation time, please refer to the relevant primary antibody reagent Instruction Manual;

(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, do not rinse the tissue surface directly with PBS buffer when rinsing the tissue sections, otherwise it may cause tissue detachment.)

F, Adding Secondary AntibodyA solution and secondary antibodyBSolution

(1)shake off excess liquid from the tissue sections,after the circle drawn by the oil pen is fully visible, Adding Secondary AntibodyASolutionto completely cover tissue ,in a wet box 37℃incubation10 minutes;

(2)Secondary AntibodyASolutionAfter incubation, ,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 sections,after the circle drawn by the oil pen is fully visible, Adding Secondary AntibodyBSolutionto completely cover tissue ,in a wet box 37℃incubation10 minutes;

(4)Secondary AntibodyBSolutionAfter incubation, ,with PBS buffer 2~3 times, then transfer to a PBS jar and soak for 2 minutes ×3 times.

G, Adding DAB Chromogen

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

(2)DAB incubation, shake off the DAB on the slides onto absorbent paper, then immerse in distilled water and rinse thoroughly for 3 minutes ×2 times to terminate the staining.

H, Counterstaining and Blue Returning

(1)After thorough rinsing with distilled water, immerse in hematoxylin staining solution for counterstaining for 8~10 seconds, and rinse clean with distilled water after counterstaining;

(2) After counterstaining, immerse in 1% lithium carbonate solution for blue returning for 8~15 seconds, and rinse clean with distilled water after blue returning.

(Note: Adjust the counterstaining time according to the intensity of different hematoxylin staining solutions, so that the cell nucleus shows light to dark blue. Over-staining or insufficient staining may affect result interpretation.)

I, Dehydration, Clearing and Mounting

(1) Shake off excess liquid from the tissue sections, immerse the tissue sections in 85% ethanol, 95% ethanol, anhydrous ethanol successively for 3 minutes;

(2)After dehydration, immerse the tissue sections in fresh xylene for 5~10 minutes;

(3) Mount the tissue sections with neutral gum and coverslips when the tissue sections become transparent.

5) Result Interpretation:

Immunohistochemical staining results should be observed and interpreted by experienced professionals under an optical microscope.

[Quality Control]

Before interpreting the staining results of the tested specimens, the staining results of a series of quality controls such as reagent controls and tissue controls should be analyzed and confirmed, compared with known positive and negative results, and referenced to previous quality-control staining results. Only when a series of controls show staining results consistent with the known results can the staining results of the tested specimens be considered valid. A complete immunohistochemical experiment should set up 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 fresh biopsy or surgical specimens, fixed, pretreated and made into paraffin-embedded sections by the same method as the tested specimens. An ideal positive tissue control should be known to contain the detected antigen in both high expression and low expression conditions, so that the experimental results can reflect different antigen expression levels in different tested specimens, especially to avoid missing low-expression cases. Normal positive tissue control staining results indicate that the tested specimen tissue is prepared correctly and the staining method is appropriate. A positive tissue control should be set up in each staining experiment. If the positive tissue control fails to show correct positive staining, the staining results of the tested specimens in this experiment should be considered invalid. (Note: Normal staining results of the positive tissue control only indicate that the experimental process and reagent performance meet the requirements, and cannot be used as an evaluation standard for the specificity of the staining results of the tested specimens.)

Negative Tissue Control should also be fixed, pretreated and made into paraffin-embedded sections by the same method as the tested specimens, and is known to have no expression of the detected antigen. A negative tissue control should be set up in each staining experiment to confirm antibody performance and provide the background of specific staining. In most cases, the tested specimen tissue sections contain different cell types, and those negative cells can serve as internal negative tissue controls. If specific positive staining appears in the negative tissue control, the staining results of the tested specimens in this experiment should be considered invalid.

Blank (or negative) Reagent Control can be performed by replacing the primary antibody with PBS buffer or IgG homologous to the primary antibody. The purpose is to evaluate the specific staining of the primary antibody, determine whether there is non-specific staining, and provide a better explanation of site-specific antigen staining. The incubation time and temperature of the blank (or negative) control reagent should be consistent with those of the primary antibody.

[Interpretation of Staining Results]

Immunohistochemical staining results should be observed and interpreted by experienced professionals under an optical microscope.

A, Immunohistochemical staining results should be based on the normal results of a series of control stainings; otherwise, the staining results of this experiment should be considered invalid.

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

C, Negative staining result (-): On the basis of normal results of a series of control stainings, no brown-yellow staining is seen in the specific cells of the tested tissue sections, indicating no expression or extremely low expression of the detected 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 accuracy of staining results.

2) A blank (or negative) reagent control, positive tissue control and negative tissue control should be set up in each staining process, and all control staining results should be normal; otherwise, the experiment is considered invalid.

3) A complete immunohistochemical experiment process includes multiple steps, including reagent selection, tissue selection, fixation and treatment, section preparation, staining and result interpretation. Tissue processing before staining can directly affect the staining effect. Inappropriate fixation, freezing, thawing, washing, drying, sectioning, or contamination by other tissues or liquids may cause false positives, inaccurate antibody localization, or false negative results. Differences in fixation and embedding methods or internal irregularities of the tissue may also cause abnormal staining results. Meanwhile, excessive or insufficient counterstaining may also affect the correct interpretation of results.

4) Interpretation of immunohistochemical staining results should be combined with pathological morphology and other data. Any interpretation of staining or its absence should be supplemented by morphology, correct controls, and other tests.

5) The reagent may have unexpected reactions in untested tissues. Due to the biological variability of antigen expression in tumor or other pathological tissues, it is impossible to completely eliminate the possibility of unexpected reactions in the tested tissues.

6) False positive results may be caused by non-immunological binding of protein 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, 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) Take 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 confirmed.

[References]

1  Wu BQ, Liu YF. Immunohistochemical tissue Pathological Diagnosis[M]. Beijing:Beijing Science and Technology Press,2007.3.

2  Dabbs(Dabbs David J) .Diagnostic Immunohistochemistrytissue Chemistry[M]. Beijing:Peking University Medical Press,2008.9.

3  Ed Harlow,David Lane. Antibody Techniques: A Laboratory Manual[M].Shen GX,Gong FL, et al., trans., Beijing:Science Press,

2002: 79-80, 105.

[Manufacturer]

Guangzhou Myabtech Biological Technology Co., Ltd.

Myabtech Biological Inc.