Calretinin Product Manual

Calretinin Instructions

Instruction Manual No.:MyM1-CR253

(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 immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections,and is used to identify the expression of Calretinin protein in tissues.

Calretinin is a calcium-binding protein with a molecular weight of 29kDa, whose gene is located on chromosome 16q22-q23. It belongs to the EF-hand protein family together with S100, and is involved in the regulation of calcium buffering, calcium sensing and cell apoptosis. It is mainly present in nervous tissues, and is also expressed in extra-neural tissues such as mesothelial cells, steroid-producing cells (adrenal cortical cells, Leydig cells, ovarian thecal cells), ovarian epithelial cells, Sertoli cells and other tissues. This antibody is mainly used for the research of epithelioid mesothelioma and adenocarcinoma. The vast majority of mesotheliomas show positive expression, localized in the nucleus and cytoplasm; only 9.8% of adenocarcinomas may show focal positive expression of calretinin, localized in the cytoplasm. Secondly, it can be used for the differential diagnosis of adrenocortical carcinoma (+) and pheochromocytoma (-), and can also be used in combination with inhibin-α for the diagnosis of ovarian sex cord-stromal tumors, in which Calretinin has a slightly higher sensitivity and inhibin-α has a slightly higher specificity. It assists in the clinical differential diagnosis of non-neoplastic mesothelial cells and tumor cells in effusions, adenocarcinoma and mesothelioma, cardiac myxoma and ovarian sex cord-stromal tumors.

(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 Calretinin protein antibody, Clone No.: MyM1-CR, Immunoglobulin isotype: 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:Improper storage of the reagent as specified above may affect its performance.)

【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 enzyme chromogen labeling the antibody is developed through redox chemical reaction, thereby 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, the chromogenic substrate is added, and 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, the samples are counterstained and mounted. The presence and expression of the antigen on the tissue sections are inferred by observing the staining under a microscope.

【Sample Requirements】

Fresh biopsy or surgical specimens should be 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 to a thickness of 3-5μm, adhere them to poly-L-lysine-coated glass 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 immunohistochemistry. If an automated immunohistochemical instrument is used for staining, adjustments should be made 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. The evaluation and verification of experimental results should 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:

The working solution of this antibody reagent does not need to be diluted 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, please 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 balsam), poly-L-lysine-coated glass 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 fresh jars of 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 (Pressure Cooker 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, put them into the retrieval solution, and 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 steam is emitted, start timing for 2 minutes from the emission of steam 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, and open the pot cover carefully (note the steam scald). Then slowly inject running water into the pot (avoid direct flushing of the tissue sections with running water);

(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 drying of the sections), 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 tissues from drying when "shaking off excess liquid from the tissue sections", otherwise the staining effect will be affected.)

C、Blocking of Endogenous Peroxidase

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

(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 distance of 2~3mm from the tissue edge. After ensuring that the entire circle encloses all the tissues, rinse gently with distilled water for 3 minutes × 2 times, and soak in PBS buffer for primary antibody incubation after rinsing off excess oil.

E、Addition of 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 the primary antibody incubation, rinse with PBS buffer for 2~3 times, and then transfer to a PBS jar for immersion washing for 2 minutes × 3 times.

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

F、Addition of 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 the secondary antibody incubation, rinse with PBS buffer for 2~3 times, and then transfer to a PBS jar for immersion washing for 2 minutes × 3 times.

G、Addition of DAB Chromogenic Solution

(1) Shake off excess liquid from the tissue sections, add the freshly prepared DAB chromogenic solution 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 for thorough rinsing 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 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, and immerse the tissue sections in 85% ethanol, 95% ethanol and anhydrous ethanol successively for 3 minutes each;

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

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

5） Result Interpretation:

Immunohistochemical staining results should be observed and interpreted by experienced professionals under an optical microscope on 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 Controlshould be fresh biopsy or surgical specimens, which are fixed, pretreated and made into paraffin-embedded sections by the same method as the tested specimens. The ideal positive tissue control should be known to contain both high 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 experiment 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 Controlshould 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 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 experiment should be considered invalid.

Blank (or Negative) Reagent Controlcan 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 at the antigen site. 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 on the stained sections.

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 site of specific cells in the tested tissue sections without background staining, indicating the presence 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 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. 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） 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 confirmed.

6） This product contains sodium azide as a preservative. Sodium azide 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 their accumulation.

【References】

1.Plattelli A et al. J Endocl 29(6):394-396, 2003.  

2.Ordonez NG et al. Hum Pathol. 35(6):697-710, 2004.

【Manufacturer】

Guangzhou Myabtech Biological Technology Co., Ltd.

Myabtech Biological Inc.