PSA Product Manual

CD3 Instruction Manual

Manual No.:MyM1-CD3253

(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 forimmunohistochemical stainingof formalin-fixed, paraffin-embedded human tissue sections, for identifying the expression of CD3 protein in tissues.

CD3 is a multi-chain transmembrane glycoprotein complex located on the surface of mature T lymphocytes, consisting of γ, δ, ε and ζ subunits. As a key component of the T-cell receptor (TCR) complex, CD3 plays a crucial role in T-cell activation and signal transduction. It is expressed in almost all mature T cells, including helper T cells (CD4+) and cytotoxic T cells (CD8+), as well as most immature T cells in the thymus. CD3 is not expressed in B cells, natural killer cells, monocytes, granulocytes or non-hematopoietic cells. As a highly specific marker for T lymphocytes, CD3 is widely used in the differential diagnosis of T-cell lymphomas/leukemias and B-cell malignancies, as well as in the evaluation of T-cell infiltration in various pathological tissues.

(Note:Interpretation of immunohistochemical staining results should be combined with pathological morphology and other immunophenotypic markers, and the staining results shall not be used as the sole diagnostic indicator.)

【Main Components】

Monoclonal mouse anti-human CD3 protein antibody, Clone No.: MyM1-CD3, 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) as a preservative, and protein stabilizers.

【Storage Conditions and Validity Period】

Store the reagent in the dark at 2-8℃ (do not freeze). Return the vial to the 2-8℃ refrigerator immediately after each use to avoid prolonged exposure to room temperature. The expiration date is printed on the outer packaging of the product (Note:Improper storage conditions may lead to antibody inactivation and affect experimental results.)

【Principle of Immunohistochemical Staining】

Immunohistochemistry is based on the principle of specific antigen-antibody binding. Antigen and antibody molecules bind specifically due to their complementary three-dimensional structures and mutual affinity. In this assay, the primary antibody (anti-CD3) binds specifically to the CD3 antigen on the surface of T lymphocytes in formalin-fixed, paraffin-embedded tissue sections. Subsequently, an HRP-labeled anti-mouse IgG polymer secondary antibody recognizes and binds to the primary antibody. Finally, a chromogenic substrate (DAB) is added, and the horseradish peroxidase (HRP) catalyzes the oxidation of DAB, producing a brown-yellow insoluble precipitate at the antigen site. The presence and distribution of CD3-positive T cells can then be observed under a light microscope.

【Sample Requirements】

Fresh biopsy or surgical specimens should be fixed immediately in 10% neutral buffered formalin for 6 to 24 hours. Prolonged fixation may mask antigenic sites and reduce staining intensity. Process the fixed tissues into paraffin blocks following standard histological procedures (dehydration, clearing, embedding). Paraffin blocks can be stored at room temperature for up to 5 years.

Cut 3-5μm thick sections from the paraffin blocks and mount them on positively charged glass slides. Bake the slides in a 58-60℃ oven for 1 hour to ensure optimal adherence. Unstained slides can be stored at room temperature (15-25℃) with desiccation. For optimal antigen preservation, it is recommended to perform immunohistochemical staining within 7 days of sectioning.

【Staining Method】

The following protocol is recommended for manual immunohistochemical staining. For automated staining platforms, adjust the protocol according to the manufacturer's instructions for the specific instrument and detection kit. A valid immunohistochemical experiment must include positive tissue controls, negative tissue controls, and reagent blanks to ensure the reliability of results. Refer to【Quality Control】and【Interpretation of Staining Results】for result evaluation.

1） Required Instruments and Equipment:

Induction cooker, stainless steel pressure cooker, digital timer, humidified incubation chamber, slide rack, staining dishes, coverslips, light microscope (40× to 200× magnification), wash bottle, absorbent paper, adjustable pipettes, etc.

2） Reagent and Material Preparation:

The ready-to-use working solution can be applied directly without dilution. For the concentrated antibody, dilute it to the working concentration using PBS (pH 7.4) or a dedicated antibody diluent at a ratio of 1:50 to 1:100 before use. Prepare other reagents (PBS, antigen retrieval solution, secondary antibody, DAB chromogen) according to their respective product instruction manuals.

Other materials required for the experiment are as follows:

PBS buffer (pH 7.2-7.4), citrate buffer antigen retrieval solution (pH 6.0), 3% hydrogen peroxide (endogenous peroxidase blocker), HRP-conjugated anti-mouse/rabbit IgG polymer, DAB chromogenic kit, hematoxylin counterstain, negative control reagent (mouse IgG), positive and negative control tissue sections, xylene or xylene substitute, graded ethanol (100%, 95%, 85%), distilled or deionized water, mounting medium (e.g., neutral balsam), positively charged slides, coverslips, etc.

3） Experimental Temperature Conditions:All incubations should be performed at room temperature (18℃-28℃).

4） Experimental Steps:

A、Deparaffinization and Rehydration

(1) Dewax sections in two changes of fresh xylene, 10 minutes each;

(2) Rehydrate sections sequentially in 100% ethanol, 95% ethanol, and 85% ethanol, 5 minutes each;

(3) Rinse gently with distilled water for 1 minute, then place in distilled water ready for antigen retrieval.

B、Antigen Retrieval (Pressure Cooker Method)

(1) Fill the pressure cooker with preheated citrate buffer (pH 6.0). Immerse the deparaffinized slides on a heat-resistant slide rack in the buffer, ensuring sections are completely submerged;

(2) Secure the lid and pressure valve. Heat on high power (1600W) until steam is vigorously released. Start timing for 2 minutes, then reduce to medium power (800W). After timing, turn off the heat and allow the pressure cooker to cool naturally for 5 minutes;

(3) Carefully move the pressure cooker to the sink. Cool the lid under running water until the pressure indicator drops. Remove the pressure valve and open the lid cautiously (avoid steam burns). Slowly add running water to the pot to reduce the temperature of the retrieval solution;

(4) When the buffer temperature is below 40℃, transfer the slide rack to distilled water. Rinse gently with distilled water for 3 minutes, twice, then proceed to endogenous peroxidase blocking.

(Note: Tissue sections are extremely susceptible to drying out after antigen retrieval. Prevent sections from drying at all subsequent steps, as this will cause non-specific background staining and invalid results.)

C、Blocking of Endogenous Peroxidase

(1) Tap off excess liquid from slides. Immediately immerse slides in 3% hydrogen peroxide solution and incubate for 10 minutes;

(2) After blocking, rinse gently with distilled water for 3 minutes, twice. Slides are now ready for circle drawing.

D、Circle Drawing

Gently shake off excess water or carefully wipe the area surrounding the tissue section. Using an immunohistochemical hydrophobic pen, draw a circle approximately 2-3mm around the tissue to create a barrier. Rinse slides gently with distilled water for 3 minutes, twice, to remove excess pen oil, then place in PBS buffer to prepare for primary antibody incubation.

E、Applying Primary Antibody or Control Reagent

(1) Tap off excess PBS from slides. Apply the diluted primary antibody working solution (or negative control IgG) to completely cover the tissue within the hydrophobic circle. Incubate in a humidified chamber for 30 minutes at room temperature;

(2) After incubation, rinse slides briefly with PBS 2-3 times, then soak in PBS for 2 minutes, three times, on a shaker.

(Note: To prevent section detachment, avoid directly squirting PBS onto the tissue surface during washing steps. Direct the stream to the slide surface adjacent to the tissue.)

F、Applying HRP-Labeled Secondary Antibody Polymer

(1) Tap off excess PBS from slides. Apply the HRP-conjugated anti-mouse/rabbit IgG polymer reagent to completely cover the tissue. Incubate in a humidified chamber for 20 minutes at room temperature;

(2) After secondary antibody incubation, rinse slides briefly with PBS 2-3 times, then soak in PBS for 2 minutes, three times, on a shaker.

G、DAB Chromogenic Reaction

(1) Tap off excess PBS from slides. Prepare DAB chromogen solution fresh according to the kit instructions. Apply the DAB solution to completely cover the tissue and incubate for 5 minutes at room temperature in the dark;

(2) Monitor the staining reaction microscopically if necessary. Terminate the reaction by tapping off the DAB solution and rinsing slides thoroughly in distilled water for 3 minutes, twice.

H、Counterstaining and Bluing

(1) After thorough rinsing with distilled water, immerse slides in Mayer's hematoxylin solution for counterstaining for 8-10 seconds. Rinse slides thoroughly with distilled water to remove excess hematoxylin;

(2) Immerse slides in 1% lithium carbonate solution for bluing for 8-15 seconds. Rinse thoroughly with distilled water to complete the bluing process.

(Note: Adjust the counterstaining time according to the batch and freshness of the hematoxylin solution. The ideal result is a light to dark blue nucleus with clear contrast against the brown DAB stain. Over-counterstaining or under-counterstaining will obscure the interpretation of specific staining.)

I、Dehydration, Clearing and Mounting

(1) Dehydrate slides sequentially in 85% ethanol, 95% ethanol, and 100% ethanol, 3 minutes each;

(2) Clear slides in two changes of fresh xylene, 5-10 minutes each, until completely transparent;

(3) Mount the slides with a drop of neutral mounting medium and a coverslip. Avoid trapping air bubbles. Allow the mounting medium to dry completely before microscopic examination.

5） Result Interpretation:

Immunohistochemical staining results must be observed and interpreted by a qualified pathologist or experienced research professional under a light microscope.

【Quality Control】

Prior to interpreting the results of test specimens, the staining patterns of all control samples (positive tissue control, negative tissue control, and reagent blank) must be evaluated and confirmed to be within the expected range. Only when all controls yield valid results can the test specimen results be considered interpretable. A comprehensive quality control strategy is essential to validate the entire experimental process, from sample preparation to staining and interpretation.

Positive Tissue Control: Use a known CD3-positive tissue (e.g., tonsil or reactive lymph node) that has been fixed and processed in parallel with the test specimens. An optimal positive control should demonstrate strong membrane staining of T lymphocytes in the paracortex. Normal staining of the positive control confirms that the tissue processing, antigen retrieval, and staining protocol are adequate. A positive control must be included in every staining run. If the positive control fails to stain correctly, the entire experiment is invalid, and results for test specimens cannot be reported. (Note:A valid positive control only verifies the technical success of the assay and does not guarantee the specificity of staining in the test specimen.)

Negative Tissue Control: Use a known CD3-negative tissue (e.g., normal colon mucosa or reactive lymph node germinal centers) processed in parallel. This control should show the absence of specific membrane staining. Additionally, non-T cells within the test specimen (e.g., B cells, histiocytes) serve as internal negative controls. Specific staining in the negative control indicates a technical error (e.g., non-specific antibody binding), rendering the experiment invalid.

Reagent Blank (Negative Control): Replace the primary antibody with an isotype-matched irrelevant mouse IgG or PBS buffer, using the same incubation conditions. This control assesses non-specific binding of the secondary antibody or other reagents. Absence of staining in the reagent blank confirms the specificity of the primary antibody-antigen interaction.

【Interpretation of Staining Results】

Interpretation must be performed by a trained professional and should always correlate with the histological morphology and the results of other relevant markers.

A、 Validity Criteria: Staining results are only interpretable if all quality control samples (positive, negative, and reagent blank) show the expected staining patterns.

B、 Positive Staining (+): In the presence of valid controls, distinct brown-yellow membrane staining is observed on the surface of lymphocytes. The staining may be linear or circumferential. This indicates the presence of the CD3 antigen, identifying the cells as T lymphocytes.

C、 Negative Staining (-): In the presence of valid controls, no specific membrane staining is observed on the target cells. This indicates the absence of detectable CD3 antigen expression.

【Limitations of the Assay】

1） Variations in specimen fixation (e.g., delayed fixation, over-fixation), tissue processing, antigen retrieval conditions, or incubation parameters can significantly impact the intensity and specificity of staining.

2） Omitting any required control or obtaining invalid results from controls invalidates the entire experiment, and test specimen results cannot be relied upon.

3） The immunohistochemical process is multi-factorial. Artifacts can arise from numerous sources, including improper slide handling (e.g., section drying, detachment), contamination, cross-reactivity with non-target antigens, or issues with counterstaining. Such artifacts may lead to false-positive, false-negative, or mislocalized staining results.

4） CD3 staining results must be interpreted in the context of morphological features and a panel of other immunomarkers (e.g., CD20, CD4, CD8). No single marker can provide a definitive diagnosis.

5） This antibody has been validated for use on formalin-fixed, paraffin-embedded human tissues. Its performance on tissues fixed with alternative fixatives (e.g., acetone, Bouin's solution) or on frozen sections has not been evaluated and cannot be guaranteed.

6） Rare cases of aberrant CD3 expression in non-T-cell malignancies have been reported in the literature. Therefore, strict correlation with morphology and other markers is mandatory.

【Precautions】

1） For Research Use Only (RUO). Not for use in diagnostic procedures, therapeutic applications, or clinical settings.

2） This product should only be used by personnel trained in immunohistochemical techniques and laboratory safety procedures.

3） This product contains biological materials. Handle all reagents as potentially biohazardous and dispose of waste in accordance with local, state, and federal regulations for biomedical waste.

4） Wear appropriate personal protective equipment (PPE), including lab coats, gloves, and eye protection, to avoid skin and eye contact with all reagents.

5） Sodium Azide Warning: This product contains sodium azide (NaN3), a toxic preservative. Sodium azide may react with lead or copper plumbing to form explosive metal azides. When disposing of solutions containing sodium azide, flush the drain with a large volume of water to prevent accumulation.

6） DAB Warning: 3,3'-Diaminobenzidine (DAB) is a potential carcinogen. Handle DAB solutions with extreme care and avoid inhalation or skin contact.

【References】

1.Alberola-Ila J, et al. Annu Rev Immunol. 1997;15:525-555.

2.von Boehmer H. Annu Rev Immunol. 1997;15:531-552.

【Manufacturer】

Guangzhou Myabtech Biological Technology Co., Ltd.

Myabtech Biological Inc.