Fixation is an essential preliminary step in IF staining in order to prevent autolysis, mitigate putrefaction, and preserve morphology while maintaining antigenicity. The ideal fixation method serves to immobilize target antigens without disturbing cellular architecture to allow antibodies maximum access to any targeted cellular components.

2022年2月23日 · After performing the necessary blocking steps, you’re now ready to stain your cells with antibodies. There are generally two principles for this: (i) direct ICC, where primary antibodies are conjugated directly with fluorophores, and (ii) indirect ICC, where a primary antibody is detected using a suitable fluorescently labeled secondary antibody.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles.

The use of primary antibodies conjugated to fluorophores, without secondary antibodies, is known as direct staining. Direct staining methods save time and allow multiplexing using antibodies raised in the same host species.

Check the product information for recommendations for each primary antibody being used. Preheat the antigen retrieval buffer (100 mM Tris, 5% [w/v] urea, pH 9.5) to 95°C. This can be done by heating the buffer in a coverglass staining jar which is placed in a water bath at 95°C.

A. Antibodies 1. Primary antibodies: purchased or your own a. if these are conjugated to a fluorochrome (e.g., FITC, PE, or other) use the DIRECT STAINING PROCEDURE b. if the primary antibody is not conjugated to a fluorochrome, use the INDIRECT STAINING PROCEDURE 2. Secondary antibodies: fluorochrome-conjugated polyclonal antibodies

primary antibody: • Short protocol, fast analysis, easy to handle. • Expensive, a labeled primary antibody for each target is needed. • A highly specific primary antibody is needed. • No signal amplification, not suitable for low expressed targets. Indirect visualization requires the use of a labeled secondary antibody:

A cocktail of antibodies may be used to stain multiple targets in the sample in a single step. Indirect staining method: In ‘indirect’ immunodetection, an unlabeled primary antibody specific for the target is then followed by a fluorophore-conjugated secondary antibody that detects the host species of the primary antibody.

Immunohistochemistry (IHC) is a technique using antibody-epitope interactions to selectively label and visualize proteins in tissue samples.

The primary antibody recognizes and binds to cell surface antigens but because there is no way to detect it’s presence on the cell surface, the cells are subsequently incubated with a fluorochrome con-jugated secondary antibody. The secondary antibody recognizes and binds to only the primary antibody molecule to provide a fluorescent reporter ...