Indirect Immunostaining Method

Deparaffinize and rehydrate tissue sections. Rinse in distilled water for 5 min. 

Antigen retrieval place sections in a Coplin jar with antigen retrieval solution of choice (e.g. 10 mM citrate acid, pH 6) and heat at 90°C 110°C (depending on tissue) in a microwave, steamer, domestic pressure cooker or autoclave. See Sect. 6.1.1. 

Washes and dilutions wash sections in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCl (PBS) for 2 x 3 min. PBS is used for all washes and dilutions. Other buffers such as Tris buffered saline (TBS) may also be used. 

Circle sections with a hydrophobic barrier pen (e.g., Dako Pen, S2002). Do not allow sections to dry for the remaining procedure. 

Blocking step incubate sections for 20 min with PBS containing 5% normal serum of species in which the secondary antibodies were raised. 

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Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

Fluorophore absorbs ultraviolet light (or violet, blue or green) and emits light of longer wavelength. Fluorophores are used in immunohistochemistry for labeling primary or secondary antibodies in direct and indirect immunostain-ing methods, respectively. They can be visualized in fluorescence microscopy using special filter sets. 

There are many minor variations of the basic method, but the constant features of all indirect immunostaining procedures are ... 

The primary remaining motive for using frozen sections in routine practice is the need for a quick examination that eliminates the time required for fixation, processing and de-waxing. Frozen tissue sections are also used when direct or indirect immunofluorescence is the detection method, in which case formalin fixation can produce weaker results. Frozen sections should be fixed with acetone (room temperature, five seconds) before storing. They are then re-processed in acetone (4 °C, 10 minutes) and then re-hydrated in buffer for five minutes before immunostaining. 

Another method takes advantage of significant differences in detection sensitivities of two immunostaining procedures. For example, the streptavidin-biotin method with biotinylated-tyr-amide amplification can detect antigens at such a low concentration of the primary antibody, that they can t be detected with the traditional indirect methods used in the subsequent immunoreac-tions (38, 39). The principles of double/multiple immunostaining techniques are summarized in Table 3. 

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