Multiple immunohistochemical staining

NegotiableUpdate on 05/06

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Nature of the Manufacturer: Producers

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Overview

Principle of multiple immunohistochemical staining: utilizing the principle of specific binding between antigens and antibodies to bind primary antibodies to target antigens in tissues or cells; Adding HRP labeled secondary antibody can specifically bind to the primary antibody, forming a complex of "antigen primary antibody HRP labeled secondary antibody". By adding casein fluorescent dye, casein is activated and covalently binds to protein residues near the antigen in the presence of HRP and *. A large amount of fluorescein deposits on the antigen antibody binding site, amplifying the signal. By repeating the above steps multiple times, using different primary antibodies and corresponding casein fluorescent dyes each time, multiple targets can be achieved

Product Details

Multiple immunohistochemical stainingExperimental Procedure

Sample preparation: including processing of paraffin sections, frozen sections, or cell slides, such as fixation, antigen repair, blocking endogenous peroxidase, etc.

Primary antibody incubation: Drop the diluted primary antibody with antibody diluent and incubate it in a dark and damp box for a certain period of time.

Secondary antibody incubation: After washing, add HRP labeled secondary antibodies of the corresponding species to the primary antibody, and incubate at room temperature in the dark.

TSA fluorescent dye staining: Add casein fluorescent dye dropwise, react at room temperature for a period of time, and then wash.

Antibody elution: Use methods such as thermal repair or antibody elution buffer to elute the previous round of antibodies for the next round of labeling.

Repeat labeling: Repeat the above steps using different primary antibodies and fluorescent dyes to achieve multiple labeling.

Double staining of cell nucleus and sealing: Use nuclear dyes such as DAPI to double stain the cell nucleus, and add anti fluorescence quenching sealing agent dropwise to seal the plate.

Microscopic examination and photography: Observe and collect images under equipment such as fluorescence microscope.

Multiple immunohistochemical stainingPrecautions

Antibody selection and optimization: High specificity and validated antibodies should be selected, and the concentration and incubation time of the antibodies should be optimized.

Epitope masking issue: Pay attention to the staining sequence of antibodies to avoid epitope masking, which can be solved by adjusting the antibody sequence or using other methods.

Experimental control settings: Positive and negative controls are set up to ensure the accuracy and reliability of the experimental results.

Control of signal amplification factor: Based on specific experimental requirements and fluorescence signal intensity, choose appropriate methods such as adding TSA enhancer to control signal amplification factor.