Protocol

Baskets for In Situ Hybridization and Immunohistochemistry

This protocol was adapted from "Immunocytochemistry," Chapter 12, in Early Development of Xenopus laevis, by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.

INTRODUCTION

For large in situ hybridization and immunohistochemistry experiments, changing solutions in vials becomes tedious, and baskets should be used. Commercially available baskets, such as 15-mm Netwell baskets (Corning), fit into 12-well tissue culture plates. Although they are expensive, they can, with extensive washing, be reused. However, these baskets are not readily adaptable to large-scale use, and their relatively shallow wells make cross-contamination between wells a real danger. This protocol describes a procedure for making homemade baskets with plastic microcentrifuge tubes and nylon mesh that are more adaptable to large-scale experiments. The baskets are narrow and deep and thus can be placed in a rack at high density. To change solutions, individual tubes or whole racks of tubes can be moved from one bath of solution to another. Because many tubes can be manipulated simultaneously, there is an enormous saving in work. It is also quite difficult to lose embryos in baskets; embryos in vials stand a good chance of being sucked into the aspirator during solution changes and lost.