Histone H3 K36M Product Manual

Histone H3 K36MInstructions

Instruction Manual No.:MyR1-H3K36M253

(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 Histone H3 K36M protein in tissues.

Chondroblastoma is composed of densely arranged chondroblasts, variable numbers of osteoclast-like giant cells, and calcified chondroid matrix. H3F3A and H3F3B are two genes encoding the histone H3.3 variant. A lysine 36 to methionine (K36M) mutation in H3F3A or H3F3B is present in 95% of chondroblastomas, predominantly in H3F3B. Studies have shown that immunohistochemical detection of the H3.3K36M mutant protein has high specificity and sensitivity in chondroblastoma, with a sensitivity of 95.6% and a specificity of 100%, which has high value in clinical practice.

(Note: 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 indicator.)

【Main Components】

Monoclonal rabbit anti-human Histone H3 K36M protein antibody, Clone No.: MyR1-H3K36M, 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 below 0℃). Return it to the 2-8℃ refrigerator immediately after each use. The validity period is indicated on the outer package 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 affinity. Based on this principle, the antibody will bind to specific antigens/proteins in the tested human tissues and cells. Subsequently, the enzyme chromogen labeled on the antibody is developed through redox chemical reactions, thereby showing the distribution of antigens 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 can catalyze 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 samples should be fixed in 10% neutral buffered formalin for 6-24 hours, and processed 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 to poly-L-lysine coated slides, and bake the slides in a constant temperature oven 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 good reproducibility of antigen distribution in tissue sections, it is recommended to complete immunohistochemical staining within 7 days.

【Staining Method】

The following are the recommended configuration and steps 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 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 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 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) Place the prepared Tris-EDTA antigen retrieval solution (pH9.0) in a pressure cooker, put 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 fire (1600W/210℃) until steam is emitted, start timing for 2 minutes from the emission of steam and switch to medium fire (800W/130℃). After the timing, 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 steadily to the sink, turn on running water to pour on the 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 cover carefully (beware of steam scalding), then slowly inject running water into the pot (avoid direct flushing of tissue sections with running water);

(4) When the temperature drops below 40℃ (water temperature 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 for 3 minutes × 2 times, 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 from drying 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 tissue sections, place the slides in peroxidase blocker (3% H2O2) quickly 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 marker pen at 2~3mm from the tissue edge. After ensuring the entire circle encloses all the tissue, rinse gently with distilled water for 3 minutes × 2 times to wash off excess ink, and soak in PBS buffer for primary antibody incubation.

E、Addition of Primary Antibody or Control Reagent

(1) Shake off excess liquid from tissue sections, add the primary antibody working solution to completely cover the tissue after the circle drawn by the marker 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, do not rinse the tissue surface directly with PBS buffer when rinsing tissue sections, otherwise it may cause section detachment.)

F、Addition of HRP-Labeled Secondary Antibody Polymer

(1) Shake off excess liquid from tissue sections, add the one-step secondary antibody reagent to completely cover the tissue after the circle drawn by the marker 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、Addition of DAB Chromogen

(1) Shake off excess liquid from 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 immerse in distilled water and rinse thoroughly for 3 minutes × 2 times to terminate staining.

H、Counterstaining and Bluing

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

(Note: Adjust the counterstaining time according to the intensity of different hematoxylin staining solutions, with the nucleus showing light blue to dark blue as appropriate. Over-staining or insufficient staining may affect the interpretation of results.)

I、Dehydration, Clearing and Mounting

(1) Shake off excess liquid from 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) When the tissue sections become transparent, mount them with neutral balsam and coverslips.

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. 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 prepared into paraffin-embedded 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 detected 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 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 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 prepared into paraffin-embedded sections by the same method as the tested specimens, and known to have no expression of the detected 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. In most cases, 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 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, 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.

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

B、 Positive Staining Result (+): On the basis of normal staining results of a series of control experiments, brown-yellow staining is seen at specific sites of specific cells in the tested tissue sections without background staining, indicating the presence and expression of the detected antigen.

C、 Negative Staining Result (-): On the basis of normal staining results of a series of control experiments, no brown-yellow staining is seen in 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 should be considered invalid.

3） A complete immunohistochemical experiment process involves 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, over-counterstaining 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, the possibility of unintended reactions in the tested tissues cannot be completely eliminated.

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 should 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 applicability of this product to 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, flush with a large amount of water to avoid accumulation.

【References】

[1] Amary M F, Berisha F, Mozela R, et al. The H3F3 K36M mutant antibody is a sensitive and specific marker for the diagnosis of chondroblastoma[J]. Histopathology, 2016.

[2] Gong L H, Zhang W, Dong R F, et al. Value of H3.3 K36M mutant protein expression in the diagnosis and differential diagnosis of chondroblastoma[J]. Chinese Journal of Pathology, 2021, 50(03):242-244.

【Manufacturer】

Guangzhou Myabtech Biotechnology Co., Ltd.

Myabtech Biological Inc.