PLAP staining protocol^§

1. Remove medium from each well and wash once with PBS. Fix cells for 30 minutes with 4% formaldehyde/PBS and wash three times with 1× Hepes buffered saline solution (8.2 g/l NaCl, 6 g/l HEPES, 0.1 g/l Na2HPO4, pH to 7.0 with NaOH).
2. Heat inactivate endogenous alkaline phosphatase by floating plates in a 65°C water bath for 45 minutes. Cool plates to room temperature.
3. Wash plates twice with alkaline phosphatase (AP) buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM Mg Cl₂) and add 0.2 ml of AP buffer containing NBT (4.5 µl/ml) and BCIP (3.5 µl/ml). Stain overnight at room temperature.
4. Remove staining solution, wash wells with PBS, and store plates at 4°C in fixative solution.

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§ Excerpted from "Gene Trapping Methods for the Identification and Functional Analysis of Cell Surface Proteins in Mice" by William C. Skarnes in: Methods in Enzymology, volume 328, edited by John N. Abelson, Jeremy Thorner, and Scott D. Emr. Copyright © 2000 by Academic Press. This material may not be reproduced, stored in a retrieval system, or transmitted in any form or by any means without the prior written permission of the publisher.