CA IX Product Manual

CA IX Instructions

Instruction No.：MyM1-CAIX253

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

【Product Name】

【Product Code and Packaging Specification】

【Intended Use】

This antibody reagent is suitable for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections to identify the expression of CA IX protein in tissues.

Carbonic Anhydrase IX is a zinc-containing metalloproteinase that catalyzes the reversible hydration of CO2 through enzyme binding to the cell membrane. It is often expressed in the gastrointestinal tract, mainly in the stomach and gallbladder. CA IX is overexpressed in a number of epithelial malignancies, including those of the uterus, cervix, lung, breast, and kidney, while not expressed in the corresponding normal tissues. The main function of CA IX is to maintain the extracellular acidic environment and promote the proliferation of tumor cells. Its sensitivity for detecting clear cell renal cell carcinoma ranges from 85% to 100%, and it can be used to distinguish clear cell renal cell carcinoma from chromophobe renal cell carcinoma, often in combination with Pax-2, Ksp-Cadherin, and CD117. CA IX shows strong diffuse and multifocal staining in urothelial carcinoma, while weak staining in renal collecting duct carcinoma, thus CA IX can distinguish between the two.

(Note：The interpretation of immunohistochemical staining results should be combined with pathological morphology and other data, and the staining results shall not be used as a diagnostic index.)

【Main Components】

Monoclonal mouse anti-human CA IX protein antibody, Clone No.：MyM1-CAIX, Immunoglobulin Classification：IgG. This product is tissue culture supernatant, containing 0.05 mol/L Tris-HCl (pH 7.2), 0.015 mol/L Sodium Azide (NaN3) and protein stabilizers.

【Storage Conditions and Validity Period】

The reagent should be stored in the dark at 2-8℃ (not lower than 0℃). Return it to the 2-8℃ refrigerator immediately after each use. The validity period is shown on the outer packaging of the product (Note：If the reagent is not stored in accordance with the above conditions, its use effect may be affected).

【Principle of Immunohistochemical Staining】

In immunology, antigen and antibody molecules specifically bind due to structural complementarity and mutual affinity. Based on this principle, the antibody will bind to the specific antigen/protein in the tested human tissues and cells. Then, the chromogen labeled with the antibody enzyme is developed through redox chemical reaction, thus showing the antigen distribution in tissues and cells.

First, this antibody specifically binds to the antigen on the tested tissue; Second, the enzyme-labeled anti-mouse/rabbit IgG polymer recognizes the antibody that has bound to the antigen; Third, add the chromogenic substrate, the horseradish peroxidase on the polymer can catalyze the decomposition of H2O2 in the DAB chromogenic solution, oxidizing benzidine to benzidine imine, thus resulting in yellow or brown-yellow staining at the antigen sites in the tissue sections; Finally, counterstain and mount the samples. Observe the color development under a microscope to infer the location and expression of the antigen on the tissue sections.

【Sample Requirements】

Fresh biopsy or surgical sample tissues are fixed with 10% neutral formalin solution for 6-24 hours, and made into wax blocks by sampling, dehydration and paraffin embedding in accordance with technical specifications. Wax blocks can be stored at room temperature for a valid period of 5 years.

Section the specimen wax blocks with 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 unstained slides can be stored at room temperature (15-25℃) (preferably with anti-oxidation treatment). For a good reproduction of antigen distribution in tissue sections, it is recommended to complete the immunohistochemical staining within 7 days.

【Staining Method】

The following are the recommended configuration and steps for manual immunohistochemical method. If an automated immunohistochemical instrument is used for staining, adjust according to different instruments and chromogenic kits. A complete immunohistochemical experiment should set up positive tissue control, negative tissue control and blank (or negative) reagent control. For the evaluation and verification of experimental results, 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 bottle, absorbent paper, pipette, etc.

2）Reagent and Material Preparation：

No dilution is required for the working solution of this antibody reagent before use. If it is a concentrate, it is recommended to dilute it into a working solution with PBS buffer or antibody diluent at a ratio of 1:100-1:200 before use. For the preparation of PBS buffer, antigen retrieval solution, secondary antibody, DAB chromogenic solution and other reagents, refer to the respective product instructions.

Other materials required for the experiment are as follows：

PBS buffer (pH7.2-7.4), Tris-EDTA antigen retrieval solution (pH9.0), endogenous peroxidase blocker, HRP-labeled anti-mouse/rabbit IgG polymer, DAB chromogenic solution, hematoxylin staining solution, blank (or negative) control reagent, positive and negative control tissue sections, xylene or xylene substitute, ethanol (anhydrous, 95%, 85%), distilled water or deionized water, mounting medium (such as neutral gum), anti-drop slides, coverslips, etc.

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

4）Experimental Steps：

A、Deparaffinization and Hydration

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

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

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

B、Antigen Retrieval (High-pressure Retrieval)

(1) Put the prepared Tris-EDTA antigen retrieval solution (pH9.0) into a pressure cooker, place the deparaffinized and hydrated tissue sections on a high-temperature resistant plastic slide rack, and put them into the retrieval solution to ensure that the tissue sections are completely immersed in the retrieval solution;

(2) Cover the pressure cooker and fasten the pressure valve, heat on high fire (1600W/210℃) until jetting, start timing for 2 minutes from the start of jetting and adjust to medium fire (800W/130℃). After the timing, turn off the induction cooker, move the pressure cooker away from the heat source stably, and cool naturally for 5 minutes;

(3) Move the pressure cooker stably to the sink, turn on running water to pour on the pot cover to cool it down until the automatic lock core falls into place, turn off the running water, remove the pressure limiting valve, open the pot cover carefully (beware of steam scald), and then slowly inject running water into the pot (avoid direct flushing of running water to the tissue sections);

(4) When the temperature drops below 40℃ (the water temperature is not hot to the touch), take out the slide rack and immerse it in distilled water quickly (avoid dry slides), rinse gently with distilled water for 3 minutes × 2 times, and soak in distilled water for blocking after rinsing.

(Note：Tissue sections are prone to drying after retrieval. In the subsequent operations, attention should be paid to preventing the tissue from drying out when "shaking off excess liquid from tissue sections", otherwise the staining effect will be affected.)

C、Blocking of Endogenous Peroxidase

(1) Shake off excess liquid from the tissue sections, place the slides quickly 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 at a position 2~3mm from the tissue edge. Ensure that the entire circle encloses all the tissue, then rinse gently with distilled water for 3 minutes × 2 times, rinse off excess oil, and soak in PBS buffer 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 30 minutes;

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

(Note：During the experiment, when rinsing tissue sections with PBS buffer, do not rinse directly on the tissue surface, otherwise it may cause the sections to fall off.)

F、Adding HRP-labeled Secondary Antibody Polymer

(1) Shake off excess liquid from the tissue sections, add the one-step secondary antibody reagent 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 20 minutes;

(2) After secondary antibody incubation, rinse with PBS buffer for 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) After DAB incubation, shake off the DAB on the slides onto absorbent paper, then put them into distilled water and rinse fully for 3 minutes × 2 times to terminate the staining.

H、Counterstaining and Blue Returning

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

(2) After counterstaining, place 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 blue to dark blue. Over-staining or insufficient staining may affect the judgment of results.)

I、Dehydration, Clearing and Mounting

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

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

(3) When the tissue sections become transparent, they can be mounted with neutral gum and coverslips.

5）Result Interpretation：

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

【Quality Control】

Before interpreting the staining results of the tested specimens, the staining results of a series of quality controls such as reagent control and tissue control should be analyzed and confirmed, compared with the known positive and negative results, and referred to the previous quality control staining results. Only when a series of controls show staining results consistent with the known ones, the staining results of the tested specimens are considered valid. A complete immunohistochemical experiment should set up positive tissue control, negative tissue control and 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, which are 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 both high expression and low expression of the tested antigen to ensure that the experimental results can reflect different antigen expression levels in different tested specimens, especially to avoid missing cases with low expression. Normal staining results of positive tissue control indicate that the tissue preparation of the tested specimen is correct and the staining method is appropriate. A positive tissue control should be set up for each staining experiment. If the positive tissue control fails to show the correct positive staining, the staining results of the tested specimens in this batch should be considered invalid. (Note：Normal staining results of positive tissue control only indicate that the experimental procedure 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 known to have no expression of the tested antigen. A negative tissue control should be set up for each staining experiment to confirm the performance of the antibody and provide a background for specific staining. Most of the time, the tissue sections of the tested specimens contain different cell types, and those negative cells can be used as internal negative tissue controls. If the negative tissue control shows specific positive staining, the staining results of the tested specimens in this batch should be considered invalid.

Blank (or Negative) Reagent Control can be selected to use PBS buffer or IgG homologous to the primary antibody to replace the primary antibody for the experiment, aiming to evaluate the specific staining of the primary antibody, judge the presence of non-specific staining, and provide a better explanation for the specific staining of antigen sites. 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 for the stained sections.

A、The immunohistochemical staining results should be based on the normal results of a series of control stainings; otherwise, the staining results of this batch 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 without background staining, indicating the presence and expression of the tested 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 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 process, 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 experiment of this batch 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. The treatment method of tissues before staining can directly affect the staining effect. Improper fixation, freezing, thawing, washing, drying, sectioning or contamination by other tissues or liquids may cause false positive, inaccurate antibody localization or false negative results. Different 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）The 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 unintended reactions in untested tissues. Due to the biological variability of antigen expression in tumors or other pathological tissues, it is impossible to completely eliminate the possibility of unintended 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）Whether this product is suitable for non-formalin fixed tissues has not been confirmed.

6）This product contains sodium azide as a preservative, which can react with lead and copper to form explosive metal azides. To prevent the formation of metal azides, rinse with a large amount of water to avoid accumulation.

【References】

[1] Leppilampi M1, Saarnio J, Karttunen TJ., et al. Carbonic anhydrase isozymes IX and XII in gastric tumors.World J Gastroenterol. 2003 Jul;9(7):1398-403.

[2] Gupta R1, Balzer B, Picken M, et al.. Diagnostic implications of transcription factor Pax 2 protein and transmembrane enzyme complex carbonic anhydrase IX immunoreactivity in adult renal epithelial neoplasms.Am J Surg Pathol. 2009 Feb;33(2):241-7.

【Manufacturer】

Guangzhou Myabtech Biological Technology Co., Ltd.

Myabtech Biological Inc.