S100 Product Manual

S100 Instructions

Instruction Manual No.:MyM1-S100253

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

【Product Name】

【Product Code and Packaging Specifications】

【Intended Use】

This antibody reagent is suitable for immunohistochemical staining of formalin-fixed, paraffin-embedded human tissue sections, for identifying the expression of S100 protein in tissues.

S100 proteins are a group of calcium-dependent regulatory proteins with EF-hand motifs, which participate in the regulation of a series of intracellular activities, including cell proliferation/differentiation, protein phosphorylation, enzyme activity, intracellular calcium homeostasis and transcription factor activity. There are three subtypes of S100 proteins: S100ao, S100a and S100b, which are expressed in melanocytes, Langerhans cells, histiocytes, chondrocytes, adipocytes, skeletal and cardiac muscle cells, Schwann cells, mammary epithelial and myoepithelial cells, salivary and sweat glands, as well as glial cells and their associated tumors. S100 proteins are mainly present in neural tissue, pituitary gland, carotid body, adrenal medulla, salivary glands and a small number of mesenchymal tissues. It assists in the clinical diagnosis and differential diagnosis of astrocytoma, oligodendroglioma, ependymoma, neuroblastoma, schwannoma, malignant melanoma and liposarcoma.

(Note: Interpretation of immunohistochemical staining results should be combined with pathological morphology and other data; the staining results are not intended as a diagnostic indicator.)

【Main Components】

Monoclonal mouse anti-human S100 protein antibody, Clone No.: MyM1-S100, 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 Shelf Life】

The reagent should be stored in the dark at 2-8℃ (not below 0℃). Return it to the 2-8℃ refrigerator immediately after each use. The shelf life is indicated on the product outer packaging (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 affinity. Based on this principle, the antibody will bind to the specific antigen/protein in the tested human tissues and cells. Subsequently, the enzyme chromogen labeled on the antibody is developed through redox chemical reactions, thereby revealing 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 horseradish peroxidase on the polymer catalyzes the decomposition of H2O2 in the DAB chromogen, oxidizing benzidine to benzidine imine, 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 in 10% neutral buffered formalin for 6-24 hours, and processed into paraffin blocks according to technical specifications for sampling, dehydration and embedding. Paraffin blocks can be stored at room temperature for a shelf life of 5 years.

Section the paraffin blocks at a thickness of 3-5μm, adhere the sections to poly-L-lysine-coated glass slides, and bake the slides in a constant temperature incubator at 58-60℃ for 1 hour for subsequent staining. Unused unstained slides can be stored at room temperature (15-25℃) (preferably with anti-oxidation treatment). For good reproduction of antigen distribution in tissue sections, it is recommended to complete immunohistochemical staining within 7 days.

【Staining Method】

The following are the recommended preparation and procedures for manual immunohistochemistry. If automated immunohistochemical instruments are used for staining, adjustments should be made according to different instruments and chromogenic kits. A complete immunohistochemical experiment should include positive tissue control, negative tissue control, and blank (or negative) reagent control. 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 humidified chamber, staining rack, staining jar, coverslips, light 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 using the concentrate, it is recommended to dilute it into a working solution with PBS buffer or antibody diluent at a ratio of 1:100 to 1:200 before use. For the preparation of PBS buffer, antigen retrieval solution, secondary antibody, DAB chromogen 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 chromogen, 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 (e.g., neutral balsam), poly-L-lysine-coated glass 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 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) Place the prepared Tris-EDTA antigen retrieval solution (pH9.0) in a pressure cooker, place the deparaffinized and hydrated tissue sections on a heat-resistant plastic slide rack, and immerse them in the retrieval solution to ensure the tissue sections are completely submerged;

(2) Cover the pressure cooker and fasten the pressure valve, heat on high heat (1600W/210℃) until steam is emitted, start timing for 2 minutes from the emission of steam and switch to medium heat (800W/130℃). After the timing is completed, turn off the induction cooker, move the pressure cooker away from the heat source steadily, and cool naturally for 5 minutes;

(3) Move the pressure cooker to the sink steadily, turn on running water to pour over the 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 cover carefully (beware of steam scald), and slowly pour 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 sections), rinse gently with distilled water twice for 3 minutes each time, and soak in distilled water for blocking after rinsing.

(Note: Tissue sections are prone to drying after retrieval. In subsequent operations, attention should be paid to preventing the tissue sections 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, place the slides in peroxidase blocker (3% H2O2) quickly and soak for 10 minutes;

(2) After blocking, rinse gently with distilled water twice for 3 minutes each time, and soak in distilled water for circling after rinsing.

D.Circling

Shake off or carefully wipe off excess liquid around the tissue sections, draw a circle around the tissue with an immunohistochemical hydrophobic pen at a distance of 2~3mm from the tissue edge. Ensure the entire circle encloses the whole tissue, then rinse gently with distilled water twice for 3 minutes each time to wash off excess hydrophobic pen solution, and soak in PBS buffer for primary antibody incubation.

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 hydrophobic pen is fully visible, and incubate at room temperature in a humidified chamber for 30 minutes;

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

(Note: During the experiment, do not rinse the tissue surface directly with PBS buffer when washing the tissue sections, 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 hydrophobic pen is fully visible, and incubate at room temperature in a humidified chamber for 20 minutes;

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

G.Addition of 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 humidified chamber for 5 minutes;

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

H.Counterstaining and Blueing

(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 blueing for 8~15 seconds, and rinse clean with distilled water after blueing.

(Note: Adjust the counterstaining time according to the intensity of different hematoxylin staining solutions, with the cell nucleus showing light blue to dark blue as appropriate. Over-staining or under-staining may affect the interpretation 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, immerse the tissue sections in fresh xylene for 5~10 minutes;

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

5) Result Interpretation:

Immunohistochemical staining results should be observed and interpreted by experienced professionals under a light 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. The staining results of the tested specimens are considered valid only when a series of controls show staining results consistent with the known ones. A complete immunohistochemical experiment should include positive tissue control, negative tissue control, and blank (or negative) reagent control to ensure the entire experimental process is accurate and controllable.

Positive Tissue Control should be fresh biopsy or surgical specimens, fixed, pretreated and processed into paraffin sections by the same method as the tested specimens. An 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 the positive tissue control indicate that the tissue preparation of the tested specimen is correct and the staining method is appropriate. A positive tissue control must 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 the 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 processed into paraffin sections by the same method as the tested specimens, and is known to have no expression of the tested antigen. A negative tissue control must be set up for each staining experiment to confirm the performance of the antibody and provide a background for specific staining. In most cases, the tissue sections of the tested specimens contain different cell types, and those negative cells can serve 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 Control can be performed by replacing the primary antibody with PBS buffer or IgG homologous to the primary antibody, aiming to evaluate the specific staining of the primary antibody, determine 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 a light 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 observed at the specific site of specific cells in the tested tissue sections with no 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 observed in specific cells in 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 should be considered invalid.

3) The complete immunohistochemical experimental process includes multiple steps, including reagent selection, tissue selection, fixation and processing, section preparation, staining and result interpretation. The processing 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 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, proper controls and other tests.

5) The reagent may produce 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) The suitability of this product 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] Banerjee SS, et al. Malignant melanoma showing smooth muscle differentiation. J Clin Pathol. 1996 Nov;49(11):950-1.

[2] Allore RJ, et al. J Biol Chem,1990,265(26):15537-43.

【Manufacturer】

Guangzhou Myabtech Biological Inc.

Myabtech Biological Inc.