Fast-Red Chromogenic Solution Product Manual

Fast-RedChromogen

Instruction Manual

Manual No.:MYF12253

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

[Product Name]

[Product No. and Packaging Specifications]

[Intended Use]

This reagent is suitable for formalin-fixed, paraffin-embedded human tissue sections immunohistochemical staining,for alkaline phosphatase (AP)labeled antibody chromogenic development.

[Main Components]

[Storage Conditions and Validity Period]

The reagent should be stored in the dark at 2 ~ 8℃(not below 0℃)store in the dark.Return to the 2 ~ 8℃ refrigerator immediately after each use.The validity period is shown on the outer packaging(Note: If the reagent is not stored under the above conditions, may affect its performance).

[Principle of Immunohistochemical Staining]

In immunology, ,antigen andantibody Composed of due to structural complementarity and affinity canspecifically bind .Based on this principle, ,the antibody binds to specific antigens in the tested Human Tissues, cells specific antigen / proteinbind .Subsequently, a redox reaction develops color with labeledantibody enzyme Chromogenchromogenic development,to show tissue and cells antigen distribution.

Firstly, ,Primary Antibodywith the tested tissue tissue antigen specifically; ;secondly, ,Alkaline Phosphatase(AP)labeled goat anti-rabbit IgGpolymer recognizes and binds the primary antibody connected to the antigen; ;thirdly, ,add chromogenic substrate; ,on the polymer APcan catalyze Fast-rednaphthol in the chromogen AS-MX phosphate decomposition to produce naphthol, ,which binds Fast-red TR to form insoluble red precipitate, ,so that tissue red staining appears at antigen sites in the tissue sections; ;finally, counterstain and mount the sample. .Observe color development under a microscope ,to infer tissue the location and expression of this antigen on the tissue section..

[Sample Requirements]

Fresh biopsy or surgical specimens are fixed with 10%neutral formalinsolutionfixed6 ~ 24 hours,sampled according to technical specifications, dehydration, paraffin embedding to make wax blocks.Wax blocks stored at room temperature are valid for 5 years.

Section the specimen wax blocks to a thickness of 3 ~ 5 μm,adhered to anti-drop treated slides,in 58 ~ 60℃°C incubator for baking slides1 hours, for staining.Unused blank slides at room temperature (15 ~ 25℃) for storage((preferably with anti-oxidation treatment)),To well reproduce tissue antigen distribution in tissue sections, , staining within 7 days is recommended.

[Staining Method]

The following are the recommended configuration and procedures for manual immunohistochemistry. If automated immunohistochemical instruments are used for staining, please adjust according to different instruments and chromogenic kits. A complete immunohistochemical experiment should set up a positive tissue control, a negative tissue control and a blank(or negative )Reagent Control,For evaluation and verification of results, refer to [Quality Control] and [Interpretation of Staining Results].

1) Required Instruments and Equipment:

Induction cooker, Pressure cooker, Timer, Incubation wet box, Staining rack, Staining jar, Coverslips, Optical microscope(40× ~ 200×), Wash bottle, Absorbent paper, Pipettes, etc..

2) Reagent and Material Preparation:

This Fast-Red chromogen should be prepared before the experiment fresh by preparing .at concentrate :Diluent= 1 : 60 ratio ,prepare sufficient Fast-RedChromogen,and mix thoroughly.

PBSBuffer, Antigen Retrieval Solution, Primary Antibody, Secondary Antibody, DABFor preparation of chromogen and other reagents, refer to each product manual.

Other materials required for the experiment are as follows:

PBSBuffer(pH 7.2 ~ 7.4), Tris-EDTAAntigen Retrieval Solution(0.01 M,pH 9.0), Endogenous Peroxidase Blocker, Primary Antibody, Secondary Antibody, DABChromogen, Hematoxylinstaining solution, Blank (or negative )Control Reagent, positive and negative control tissue sections, xylene or xylene substitute, ethanol (anhydrous, 95%, 85%), distilled or deionized water, mounting medium(such as neutral gum), anti-drop 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;

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

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

B, Antigen Retrieval:Please refer to the relevant primary antibody reagent Instruction Manual; 

C, Blocking of Endogenous Peroxidase

(1) Shake off excess liquid from the tissue sections, place the slides 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. The circle should be 2~3mm from the tissue edge; after ensuring the circle encloses all tissue, rinse gently with distilled water for 3 minutes ×2 times, and after rinsing off excess oil, soak in PBSBuffer 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 ,For incubation time, please refer to the relevant primary antibody reagent Instruction Manual;

(2) After primary antibody incubation, rinse with PBS buffer 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 the tissue sections, otherwise it may cause tissue detachment.)

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 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)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 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 to dark blue. Over-staining or insufficient staining may affect result interpretation.)

I, Dehydration, Clearing and Mounting

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

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

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

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. Only when a series of controls show staining results consistent with the known results can the staining results of the tested specimens be considered valid. A complete immunohistochemical experiment should set up a positive tissue control, a negative tissue control and a blank(or negative )Reagent Control,to ensure the entire experimental process is accurate and controllable.

Positive Tissue Control should be fresh fresh biopsy or surgical specimens,fixed by the same method as the tested specimens, pretreated and made into paraffin sections .An ideal Positive Tissue Controlshould contain known two detected antigen expression results ,to ensure results reflect different antigen expression levels in different specimens,especially to avoid missing low expression cases.Normal Positive Tissue Controlstaining indicates the tested specimen tissue is prepared correctly,and the staining method is appropriate.Each staining experiment requires Positive Tissue Control.If Positive Tissue Control fails to show correct positive staining, ,the staining results of the tested specimens should be considered invalid.(Note: Normal staining results of the positive tissue control only indicate that the experimental process 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 by the same method as the tested specimens, pretreated and made into paraffin sections ,and is known to have no two detected antigen expression .Each staining experiment requires Negative Tissue Control,to confirm antibody performance,and provide background for specific staining.In most cases, the tested specimen tissue sections contain different cell types, and those negative cells can serve as internal negative tissue controls. If specific positive staining appears in the negative tissue control, 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 PBSBufferor IgG homologous to the primary antibody mixed IgGreplace the primary antibody in the experiment,to evaluate specific staining of the primary antibody,determine non-specific staining,and better explain site-specific antigen staining.Blank (or negative )Incubation time and temperature of control reagent should match 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 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 control staining results, ,tested tissue specific sites of specific cells in tissue sections respectively show brown-yellow and red staining, ,or a dual-stained cell is observed, ,with no background staining, ,indicating two expression of the detected antigen.(Note: Both colors must be observed simultaneously at the same focus.)

C, Negative staining result(-):On the basis of normal control staining results, ,tested tissue no staining is seen in specific cells of tissue sections with brown-yellow and red staining, ,or only brown-yellow or red staining, ,indicating one or both detected antigens are not expressed or expressed at extremely low levels.

[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 Controland Negative Tissue Control,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. Tissue processing before staining can directly affect the staining effect. Inappropriate fixation, freezing, thawing, washing, drying, sectioning, or contamination by other tissues or liquids may cause false positives, inaccurate antibody localization, or false negative results. Differences in fixation and embedding methods or internal irregularities of the tissue may also cause abnormal staining results. Meanwhile, excessive or insufficient counterstaining may also 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, correct controls, and other tests.

5) The reagent may have unexpected reactions in untested tissues. Due to the biological variability of antigen expression in tumor or other pathological tissues, it is impossible to completely eliminate the possibility of unexpected 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 caused by .

[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) The suitability of this product for non-formalin fixed tissues has not been confirmed.

[References]

1 Xu Y. IHC dual staining for screening patients suitable for PD-L1 inhibitor therapy in non-small cell lung cancer[D].Huazhong Agricultural University,2021.DOI:10.27158/d.cnki.ghznu.2021.000612.

2 Han X,Song GX,Wang M, et al..Experience with dual IHC staining using same-species antibodies[J].J Clin Exp Pathol,2022,38(12):1518-1519.DOI:10.13315/j.cnki.cjcep.2022.12.023.

3 Cui BP,Gao CX,Xiong CQ, et al..A simple , reliable new dual IHC labeling method[J].Chin Pharmacol Bull,2015,31(03):436-441.

4 Si C,Zeng Z,Zhan CZ.Application of IHC dual staining in lymphoma subtyping[J].China Med Herald,2017,29(21):79-81.

5 Shi L,Zhu XL,Yang HS, et al..IHC dual staining in lung and prostate tumor biopsiestissue should with [J].J Clin Med Lit (Electronic),2017,4(08):1388-1390.DOI:10.16281/j.cnki.jocml.2017.08.002.

[Manufacturer]

Myabtech Biological Inc.

Myabtech Biological Inc.