Microarray immunostaining

The staining procedure for carbohydrate microarrays is basically identical to the routine procedure for immunohistology. Immunostaining steps of carbohydrate arrays are listed below (see Notes 4 and 6 and Fig. 3). 

Nonaka and Chiriboga studied basal cell differentiation in 108 cases of stage 1 primary lung adenocarcinoma by immunohistochemistry using tissue microarrays with immunostains for p63, TTF-1,... 

The methods described herein encompass (1) preparation of a whole cell lysate from either cell culture or tissue samples, (2) protein lysate microarray printing, (3) immunostaining, and (4) microarray spot analysis. 

Each microarray is probed with a single primary antibody directed against the protein of interest. Microarray slides used for immunostaining should be blocked before staining. Microarray slides used for Sypro Ruby protein staining do not require blocking. 

Immunostaining requires the use of control slides for determining background staining due to secondary antibody alone. Each species-specific secondary antibody should be used to stain an individual microarray. Therefore, for any immunostaining run, one slide must be used as a negahve (secondary antibody alone) control slide. The total number of slides to be stained is determined by the number and species of primary antibodies. For example, if three anti-rabbit primary antibodies and one mouse primary antibody are selected, a total of 6 microarray slides will be needed (4 antibodies + 2 controls). 

Figure 3.5 Schematic representation of tissue microarray (TMA) fabrication. The tissue cylinders are taken by a sampling needle and embedded into the acceptor block, followed by immunostaining and evaluation of staining intensity.

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