This diluent is a ready-to-use working solution, specially formulated for diluting primary and secondary antibodies in immunoassays including immunohistochemistry (IHC) and immunocytochemistry (ICC). It contains stabilizers and protective ingredients to maintain the activity of diluted antibodies, accelerate the binding between primary antibodies and antigens, and significantly save experimental time. Antibody working solutions prepared with this diluent can be stored at 4°C and reused for no less than one week, helping conserve valuable antibody reagents.
Replace overnight incubation with just 1 hour; one-step incubation with no blocking required.
Compatibility: This diluent is also suitable for 4°C overnight incubation and is universally applicable to both primary and secondary antibodies.
Main Ingredients: Proprietary buffer system, protein stabilizer, preservative.
Reference Immunohistochemistry (IHC) Experimental Procedures:
Step
| Operation
| Key Details and Recommendations
| Deparaffinization and Hydration
| Deparaffinize sections with xylene, followed by gradient ethanol hydration to distilled water.
| Ensure complete deparaffinization; otherwise, uneven staining or high background is likely to occur.
| Antigen Retrieval
| Perform heat-induced antigen retrieval with an appropriate buffer (e.g., Citrate Buffer, Cat.No. C752119 or EDTA Buffer, Cat.No. E745530) according to the requirements of the primary antibody.
| Allow the sections to cool naturally to room temperature after retrieval before proceeding to the next steps.
| Endogenous Enzyme Blocking
| Incubate sections with Endogenous Peroxidase Blocking Solution (Cat.No. E752115) for 10–30 minutes to eliminate endogenous peroxidase activity.
| / | Primary Antibody Incubation
| Prepare the primary antibody working solution with this diluent at the initial concentration recommended in the antibody instructions, then add dropwise to the tissue section.
| Choose one of the two incubation protocols:A. Rapid protocol: Incubate at 37°C for 1–2 hours; avoid drying out of sections at all times.B. Conventional protocol: Incubate overnight at 4°C, then wash thoroughly 3 times with TBST.
| Secondary Antibody Incubation
| Dilute HRP-conjugated secondary antibody at a ratio of 1:200 to 1:500 using this diluent, and apply dropwise to the sections.
| Incubate at room temperature or 37°C for 30 minutes to 1 hour. Afterwards, wash thoroughly 3 times with TBST.
| Color Development & Counterstaining
| Add DAB (Cat. No.: D405772) or other chromogenic substrates, and control the color development time under a microscope.
| The DAB color development time should be flexibly controlled. Terminate the reaction once the target signal becomes clear with low background staining.
| Nuclear Staining
| Terminate the reaction with tap water, then perform nuclear counterstaining with hematoxylin (Cat.No.: H1507743) for 3–5 minutes.
| If the target protein is expressed in the cell nucleus, hematoxylin staining can be omitted or the staining time can be shortened appropriately.
| Mounting
| After dehydration and clearing of the sections, mount slides with non-toxic neutral balsam (Cat.No.: A1508972) for long-term preservation.
| Conventional neutral balsam contains xylene; the use of non-toxic neutral balsam is recommended. |
|
Incubation Time Selection
You can appropriately adjust the incubation time according to the expression level of the target protein:
Protocol
| Primary Antibody Incubation
| Secondary Antibody Incubation
| Application Scenario
| Protocol 1
| 37°C, 1–2 h
| Room temperature / 37°C, 30 min–1 h
| High-abundance proteins with relatively strong non-specific binding; results required on the same day.
| Protocol 2
| 4°C, Overnight
| Room temperature / 37°C, 30 min–1 h
| Detection of low-abundance targets; pursuit of high signal-to-noise ratio.
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Precautions:
1. Scope of application: This product is only for scientific research use by professionals. It shall not be used for clinical diagnosis, treatment, or any food and pharmaceutical applications.
2. Antibody recovery: To prolong the service life of recovered antibodies, it is recommended to perform primary antibody incubation at 4°C.
3. Key operational notes.
4. Antibody dilution: Always follow the starting dilution concentration recommended in the antibody manufacturer’s instructions for primary antibody preparation. The general starting dilution range for secondary antibodies is 1:200 to 1:500.
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