Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy 

HAF © 2013 

Vol. IV, No. 1s   ISSN: 2283-3927 

   

 

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Keywords 

FXR1, Western blot, 

Immunohistochemistry, Oral 

melanoma, Dog 

 

CORRESPONDING AUTHOR 

Laura Nordio 

laura.nordio@unimi.it 

 

JOURNAL HOME PAGE 

riviste.unimi.it/index.php/haf 

Validation of anti-FXR1 antibodies in the  

canine species and application to an  

immunohistochemical study of canine  

oral melanomas 

Laura Nordio1*, Andreia F. T. Marques1 

 

 

 

 

 

1University of Milan, Department of Veterinary Medicine, Italy  

 

Abstract 

FXR1 (Fragile X mental retardation-related protein 1) is a cytoplasmic RNA binding protein (Siomi et al., 

1995), which genetic expression has been related to metastatic potential in human melanoma. The 

aims of the present study were to validate two commercially available clones of polyclonal anti-human 

FXR1 antibody in dogs and to use them to investigate FXR1 expression in a group of canine oral 

melanomas. Anti-FXR1 antibody was never validated before in the canine species. 

Two different commercially available polyclonal anti-FXR1 antibodies (raised in goat and rabbit, 

respectively) were used. FXR1 protein in canine serum was identified by western blot after SDS-PAGE, 

using human serum as control. FXR1 immunohistochemical expression was tested in a series of normal 

tissues, that are expected to express FXR1, and in 31 cases of oral melanomas. The final 

immunohistochemical protocol used heat-induced unmasking and overnight incubation. 

FXR1 protein bands in canine serum were detected by validated antibodies. The rabbit antibody 

specifically identified a band around 65 kDa (Figure 1A), whereas the goat antibody reacted also with 

other not specific bands. FXR1 immunohistochemical staining was positive in all tested organs, with 

different levels of expression. FXR1 was also expressed in 31/31 tested melanomas, with variable 

intensity and percentage of positive cells (Figure 1B). Equal results were achieved with the two 

antibodies in 8 cases of melanoma, whereas there were variable differences in 22, and one case 

stained only with goat antibody. The rabbit antibody gave less background staining. This study 

validated anti-FXR1 antibodies for use in the canine species. This protein was identified in several 

normal tissues, as well as in the tested neoplasms. Significance of different level of expression is 

undergoing evaluation with further studies. 

 

Acknowledgments: Francesca Genova, Valentina Serra, Maria Lina Longeri, Chiara Bazzocchi, Cristina 

Lecchi, Fabrizio Ceciliani, Chiara Giudice.  

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en


Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy 

HAF © 2013 

Vol. IV, No. 1s   ISSN: 2283-3927 

 

 

 

 

 

 

 

 

 

 

 
 

Figure 1. A) Western blot, rabbit anti-FXR1 antibody, positive bands around 65 kDa. B) Immunohistochemistry, oral 

melanoma, cytoplasmic positivity of neoplastic cells to FXR1. Rabbit anti-FXR1 antibody, AEC chromogen, 40x. 

 

References 

Siomi, M.C., Siomi, H., Sauer, W.H., Srinivasan, S., Nussbaum, R.L., Dreyfuss, G., 1995. FXR1, an autosomal homolog 
of the fragile X mental retardation gene. EMBO J. 14: 2401-2408.ormation. Journal of Microbiological Methods. 40, 
175–179. 

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en