Automation of APAAP immunocytochemical technique.

Abstract

SUMMARY A tissue processing instrument (the Histokinette) was modified by the addition of an electronic timing device which allows an immunocytochemical staining technique (the APAAP method) to be performed as a semiautomated procedure. After incubation with primary monoclonal antibodies (applied by hand) slides (up to 72 in a batch) are placed in racks and cycled through tanks of reagents, comprising anti-mouse Ig followed by APAAP complexes with intervening timed draining and washing stages. This semiautomated process gave consistent staining results and offered considerable savings in time compared with conventional methods. The same reagent baths were used over four months on an almost daily basis without deterioration in staining intensity, and consequently the calculated overall cost of the staining procedure was less than if the reagents had been applied by hand and then discarded. The machine is now into its eleventh month of operation; the reagents have been changed twice.It is suggested that this approach, because of savings in time and increased consistency, may be an attractive technique for the routine immunocytochemical staining of slides, and that the nature ofthe APAAP method is particularly suitable for automation as the necessary reagents can be produced at low cost.The APAAP immunoalkaline phosphatase technique' (table 1) has been widely used for staining histological and haematological specimens; the advantages of the method have been reviewed elsewhere.2 APAAP staining, however, entails considerable technical time, and results depend, at least in part, on the skill and care of the person performing the staining. There would therefore be considerable benefits in a technique which automated at least part of the procedure. The manual technique is also inherently wasteful in that each of the reagents (usually in a volume of 50-100 p1) is pipetted directly on to the slides and then washed away in buffer at the end of each incubation period. As the antimouse Ig and APAAP are applied at saturating concentration and are not exhausted after a single incubation, there is unnecessary wastage, which would be avoided if the reagents were reused.For these reasons we attempted to develop an automated procedure for performing the APAAP procedure based on incubation of slides in tanks of reagents which can be used repeatedly over a period of time. Incubation with primary antibodies is the most difficult step in the procedure to automate as it is common practice to use several different reagents in a staining run, and primary antibodies are often only

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