Application Notes
Multiplex immunohistochemical, also known as Tyramide dignal amplification (TSA), is an enzymatic assay that uses horseradish peroxidase (HRP) for
high-density in situ labelling of target proteins or nucleic acids. It is a class of enzymatic assays that use horseradish peroxidase (HRP) for high-density
in situ labelling of target proteins or nucleic acids. It is based on multiple cis-immunostaining with tyramide signal amplification, which allows the
detection of multiple targets in situ in cell or tissue samples, and elucidation of their interaction mechanisms through the study of their combinations
and positional relationships.
General Notes
1. This kit is suitable for use in mIHC (Multiplex Immunohistochemistry). Creative Diagnistics offers a wide range of multiplex fluorescence kits for simultaneous detection of up to seven targets to maximise customer satisfaction
2. Sample Requirements
Tissue samples should be fresh biopsy or surgical tissue samples, fixed in 10% neutral buffered formalin for 8 - 24 hours. Sample collection, dehydration, and paraffin embedding should be performed according to pathological technical specifications to make paraffin blocks. It is recommended that the tissue section thickness be 3 - 4 μm, and use anti-slip slides.
3. Reagent Preparation (* Prepare immediately before use.)
Fluorescence Staining Solution: Prepared by mixing TSA Dye and Amplification Solution at a ratio of 1:100.
DAPI Staining Solution: Prepared by mixing DAPI Concentrate and PBS Buffer at a ratio of 1:100.
4. Test Conditions(temperature): 18 - 25°C
5. General protocol
5.1. Deparaffinization and Hydration
Place paraffin sections in fresh xylene and soak twice for 10 minutes each time.
After removing the excess liquid, place them in anhydrous ethanol and soak twice for 5 minutes each time.
After removing the excess liquid, place them in 95% ethanol and soak for 5 minutes.
After removing the excess liquid, place them in 85% ethanol and soak for 5 minutes.
Rinse with tap water three times for 3 minutes each time.
5.2 Antigen Retrieval
Boil for 3 minutes in a pressure cooker (alternatively, use a microwave oven at high power for 3 - 5 minutes until boiling, then adjust the power to low and heat for 15 - 20 minutes to ensure the liquid does not boil). Allow to cool naturally, soak in purified water twice for 3 minutes each time, and soak in the washing solution once for 3 minutes. During antigen retrieval, the amount of retrieval solution must ensure that the sections are always immersed in the liquid
5.3 Add Peroxidase Blocking Agent
Rinse the antigen-retrieved sections with purified water three times for 3 minutes each time. Circle the area of the tissue to be tested on the slide with an oil pen. Rinse with the washing solution once for 3 minutes.
Remove the washing solution and add 100 μl of peroxidase blocking agent within the area circled by the oil pen (the amount of reagent depends on the size of the section, preferably to completely cover the tissue section). Incubate at room temperature for 10 minutes. Rinse with the washing solution three times for 3 minutes each time.
5.4 Add Primary Antibody
Remove the washing solution and add 100 μl of primary antibody (the amount of reagent depends on the size of the section, preferably to completely cover the tissue section). Incubate at room temperature for 40 - 60 minutes. Rinse with the washing solution three times for 3 minutes each time.
5.5 Add Enzyme-Labeled Polymer
Remove the washing solution and add 100 μl of enzyme-labeled polymer (the amount of reagent depends on the size of the section, preferably to completely cover the tissue section). Incubate at room temperature for 20 - 30 minutes. Rinse with the washing solution three times for 3 minutes each time.
5.6 Fluorescence Development
Remove the washing solution and add 100 μl of the prepared fluorescence staining solution onto the slide (the amount of reagent depends on the size of the section, preferably to completely cover the tissue section). Incubate at room temperature for 5 - 10 minutes. Rinse with the washing solution three times for 3 minutes each time.
5.7 Microwave Treatment (to remove primary and secondary antibodies)
Remove the washing solution. Boil the antigen retrieval solution and place the slide in the antigen retrieval box. Adjust the microwave power to low and continue the retrieval for 15 - 20 minutes (ensure that the tissue is immersed in the liquid throughout the process). Allow to cool naturally at room temperature (15 - 30 minutes). Rinse the slide with purified water and soak the slide in the washing solution for 2 minutes.
5.8 Multiple Rounds of Staining
Remove the washing solution and continue with subsequent indicator staining. Repeat steps 3 - 7 until all antigens are labeled.
5.9 DAPI Staining
Remove the washing solution and add 100 μl of the prepared DAPI staining solution to the sample area on the slide (the amount of reagent depends on the size of the section, preferably to completely cover the tissue section). Incubate at room temperature for 5 - 10 minutes. Rinse with deionized water three times for 3 minutes each time.
5.10 Mounting
Add anti-fluorescence quenching mounting medium onto the slide for mounting, and fix the coverslip with nail polish.
6. Precautions
6.1. Read the instruction manual carefully before starting the experiment.
6.2. Before use, make sure that the laboratory's fluorescence microscope or fluorescence scanner and other observation equipment match the excitation/absorption wavelength parameters of the fluorescent dyes.
6.3. This reagent must be used by trained professionals within the expiration date. If leakage, contamination, or deterioration is found, do not use it.
6.4. If the components of this reagent are mixed with products from other companies, abnormal staining may occur.
6.5. Appropriate protective measures should be taken when using this reagent to avoid contact with the skin and eyes. Do not suck the reagent by mouth. If the skin, mucous membranes, or other parts of the body come into contact with the reagent, rinse immediately with a large amount of water.
6.6. Positive control slides and blank control reagents must be used simultaneously during each staining process; otherwise, the results are not acceptable.
6.7. Antigen retrieval, incubation time, temperature conditions, or the application of other methods may lead to incorrect results.
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