I. Introduction:

The primary technical principle of multiplex immunohistochemistry (mIHC) is derived from TSA technology. TSA, which stands for tyramide signal amplification, is an enzymatic detection method that utilizes horseradish peroxidase (HRP) for high-density in situ labeling of target proteins or nucleic acids. The method of multiplex fluorescent staining using this technique involves the use of HRP conjugated to secondary antibodies (rather than direct conjugation of fluorophores) to catalyze the subsequent addition of non-active fluorophores to the system. These fluorophores are activated under the action of HRP and hydrogen peroxide and are covalently coupled to the tyrosine residues of adjacent proteins, resulting in stable binding of the protein samples to the fluorophores. Subsequently, microwave treatment is applied to remove non-covalently bound antibodies, while the covalently bound fluorophores remain on the sample. The process is repeated with a different primary antibody for the next round of incubation. After all antibodies have been incubated and the fluorophores have been successfully bound, the final detection is performed.

 

Due to the multiple microwave treatments involved, it seems that only paraffin-embedded sections can withstand such a process. Researchers with frozen samples have previously been unable to use this technique, as frozen sections cannot endure even a single round of heat treatment. However, with the advancement of technology, this challenge is gradually being overcome. To enable more researchers to utilize mIHC, Absin has introduced an antibody stripping buffer (abs994). Let's now explore how to conduct multicolor experiments on frozen sections.

 

II. Suggested Operating Procedures:

 

1. Sample Preparation

1) Fixation and Embedding: Obtain fresh tissue, fix with 4% paraformaldehyde at low temperature, wash three times with PBS buffer for 15 minutes each; dehydrate with 30% sucrose, and embed in OCT;
2) Sectioning: Using a cryostat, cut the tissue into 3-5μm sections (for tissues stored at -80℃, transfer to a -20℃ freezer for about 15 minutes to equilibrate before sectioning), adhere to adhesive-coated slides, air-dry at room temperature for 30-60 minutes. Wash the sections in PBS three times for 5 minutes each to remove OCT;
3) Quenching Endogenous Peroxidase: Add 3% H₂O₂, incubate at room temperature for 10-30 minutes, wash with PBS for 5 minutes × 3 times.

2. Multicolor Experiment
1) Blocking: Add blocking solution, incubate at room temperature for 30 minutes, remove the blocking solution;
2) Primary Antibody Incubation: Discard the blocking solution, add diluted primary antibody working solution, incubate overnight at 4°C or for 1-2 hours at 37℃ in a humidified chamber in the dark (a small amount of water can be added to the chamber to prevent drying during antibody incubation), wash with PBS for 5 minutes × 3 times;
3) Secondary Antibody Incubation: Add HRP-conjugated secondary antibody corresponding to the primary antibody species to cover the tissue, incubate at room temperature for 20-50 minutes in the dark, wash with PBS for 5 minutes × 3 times;
4) Dye Incubation: Add freshly prepared 1× TSA dye working solution (diluted 1:100 with signal amplification solution), react for 2-15 minutes, wash with PBS for 5 minutes × 3 times;
5) Antibody Stripping: Add an appropriate amount of antibody stripping buffer (abs994) pre-warmed to 37℃ and completely dissolved to cover the sample, remove after 10 seconds, add antibody stripping buffer again to cover the entire sample and slightly bulge, incubate in a humidified chamber at 37℃ for 5-20 minutes (adjust stripping temperature and time according to section thickness and primary antibody type, stripping difficulty: structural membrane proteins and cytoskeletal proteins > cytoplasmic proteins > nuclear proteins), wash with PBS for 5 minutes × 3 times;
6) Repeat the process from [1 Blocking → 5 Antibody] stripping until all target staining is completed;
7) Add 1× DAPI working solution to the sample, cover the sample area, incubate at room temperature for 10 minutes, wash with PBS for 5 minutes × 3 times;
8) Add anti-fade mounting medium, cover with a cover slip to avoid bubbles;
9) Scan and image, analyze data.

 

III. Precautions:

 

1. If certain antibodies are difficult to strip, it is recommended to reduce the dilution ratio of the primary antibody or place that target in the last round of staining;
2. The antibody stripping buffer should be adjusted for incubation time and stripping temperature according to the actual situation. If the time is too long, it may damage the antigen target and nuclear morphology;
3. Please fix the sample before embedding. Post-embedding fixation may not withstand multiple labeling and stripping;
4. For frozen sections, control the section thickness to no more than 5um. Thicker samples may affect staining results.

 

Although the antibody stripping buffer solves the problem of heat repair in frozen sections, multiple rounds of stripping may still result in section detachment. Use blank sections to test the tolerance of multiple antibody stripping to determine the final number of staining targets.

 

Catalog No.	Product Name	Specification
abs50012	Absin 4-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T
abs50028	Absin 4-Color IHC Kit(Anti-Rabbit Secondary Antibody)	20T
abs50013	Absin 5-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T
abs50029	Absin 5-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T
abs50014	Absin 6-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T
abs50030	Absin 6-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T
abs50038	Absin 7-Color IHC Kit (plus) (Anti-Rabbit Secondary Antibody)	20T
abs994	Antibody eluent (for mIHC)	30ml

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