[Histonet] GUS staining on parifin thin sections, or infiltration os stain into developing embryos

[John Kiernan]

The following three papers indicate that attention to technical details is important in the detection of beta-glucuronidase as a reporter gene. 
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1. Caissard JC, Guivarch A, Rembur J, Azmi A, Chriqui D (1994) Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay. Transgenic Research 3: 176-181.

2. Tague BW, Gallant P, Goodman HM (1997) Expression analysis of an Arabidopsis C2H2 zinc finger protein gene. Plant Molecular Biology 32: 785-796.
 
3. Guivarch A, Caissard JC, Azmi A, Elmayan T, Chriqui D, Tepfer M   1996
In situ detection of expression of the gus reporter gene transgenic plants: Ten years of blue genes 
Transgenic Research 5 (5): 281-288 SEP 1996 
Abstract: 
Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on the gus (uidA) gene of Escherichia coli, which encodes a beta-glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles using gus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations, based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/or in situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels. 
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X-gal, X-gluc etc are nicknames for bromo-chloro-indoxyl glycosides, which can generate insoluble indigo-like pigments after enzyme-catalysed hydrolysis followed immediately by oxidation. The same X-* substrates are used also in methods that trap the products as insoluble azo dyes or as formazans. These methods are from the Golden Age of enzyme activity histochemistry (1960s and 1970s). 
 
A recent review of indigogenic substrates (Indigogenic substrates for the detection and localization of enzymes. Biotechnic & Histochemistry 82, 73-103, 2007) covers many sources of false-positive and false-negative artifacts. 
 
John Kiernan
Anatomy, UWO
London, Canada
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----- Original Message -----
From: Michelle Jamison <mjamison <@t> caissonlabs.com>
Date: Thursday, January 7, 2010 18:30
Subject: [Histonet] GUS staining on parifin thin sections, or infiltration os stain into developing embryos
To: histonet <@t> lists.utsouthwestern.edu

> Hi All
> I have been trying to stain for GUS expression (X-Gluc) in 
> developing seeds of Arabidopsis.  I have good expression on 
> my positive control (a "housekeeping gene) except for the 
> developing embryos!  And forget about those samples with 
> the specific gene in the embryos.  One of the things I will 
> be trying is thin sectioning then staining.  Possibly the 
> enzyme will be compromised?  
> Does anyone have experience with this sort of thing? 
> Please let me know your thoughts and or experiences.
> Thank you
> Michelle
> 
>  
> 
> Michelle Jamison
> Cytology 
> Caisson labs
> 1740 Research Parkway
> North Logan Utah
> 84341
> 435.755.7617 ext 109