Histochemical Staining of β-Glucuronidase and Its Spatial Quantification
Microscope images of plant specimens showing expression of GUS markers, besides being very beautiful, provide useful information regarding various biological processes. However, the information extracted from these images is often purely qualitative, and in many publications is not subjected to quantification. Here, we describe a very simple quantification method for GUS histochemical staining that enables detection of subtle differences in gene expression at cellular, tissue, or organ level. The quantification method described is based on the freely available image analysis software ImageJ that is widely used by the scientific community. We exemplify the method by quantifying small and precise changes (at the cellular level) as well as broad changes (at the organ level) in the expression of two previously published reporter lines, such as the pPILS2::GUS and pPILS5::GUS. The method presented here represents an easy tool for converting visual information from GUS histochemical staining images into quantifiable data and is of general importance for plant biologists performing GUS activity-based evaluation of reporter genes.
Key wordsGUS staining Reporter gene expression PILS Auxin Quantification ImageJ
We thank Esteban Fernandez for his online posts to scientific questions and David Whittaker for help with the manuscript. This work was supported by the Vienna Science and Technology Fund (WWTF) (Vienna Research Group), Austrian Science Fund (FWF) (P26568-B16; P26591-B16 (to J.K.-V.) and T-728-B16 (to E.F.)), and the European Research Council (ERC) (Starting Grant 639478-AuxinER (to J.K.-V.)).
- 4.Jefferson RA (1988) Plant reporter genes: the GUS gene fusion system. In: Setlow JK (ed) Genetic engineering, vol 10. Plenum Publishing Corporation, New YorkGoogle Scholar
- 6.Abramoff MDM, Paulo J, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11(7):36–42Google Scholar
- 7.Barbez E, Kubes M, Rolcik J, Beziat C, Pencik A, Wang B, Rosquete MR, Zhu J, Dobrev PI, Lee Y, Zazimalova E, Petrasek J, Geisler M, Friml J, Kleine-Vehn J (2012) A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature 485(7396):119–122CrossRefPubMedGoogle Scholar