Immunostaining labeled

Figure 7.9 Appropriate antigen retrieval and immunostaining of peptide controls and tissue sections, stained for HER2. The tissue section on the left has an island of 3+ HER2 tumor, toward the top of the tissue section. The tissue section on the right does not express HER2. Identifying information on the label was removed. See color insert.

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. 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

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. 

Pizzolo, G. and Chilosi, M. (1984) Double immunostaining of lymph node sections by monoclonal antibodies using phycoerythrin labeling and haptenated reagents. Am. J. Clin. Pathol. 82,44-47. 

FIGURE 11.2 Inhibitory effect of halichlorine on NF- activation in BAECs. (A) BAECs were preincubated with or without halichlorine ( ) for 2h and then exposed to LPS (3 ig/ml) for 3h. Next, cells were collected and then analyzed by RT-PCR. Agarose gel electrophoresis demonstrates mRNA encoding GAPDH, VCAM-1, ICAM-1, and E-selectin. (B) BAECs were preincubated with or without halichlorine ( ) for 2h and then exposed to LPS (3pg/ml) for lh. The p65 subunit of NF- was immunostained with Alexa Flour 568-labeled antibody (red) and nuclei was stained with DAPI (blue). This figure is sited from Tsubosaka et al., 2010b, and is permitted for use by the publisher. 

FIGURE 4.1. Effect of antibody (PC 10) concentration on the immunostaining of PCNA antigen in rectal mucosal proliferating cells. (A) The antibody was used at a concentration of 1 100. (B) The antibody was used at a concentration of 1 400. Note the increased labeling, especially in the upper crypt, when antibody concentration of 1 100 was used. Arrows indicate the upper limit of labeled cells. Reproduced, with permission, from Holt et al. (1997). Copyright 1997 American Association for Cancer Research. 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

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