Gluten proteins and their impact in the quality of wheat based food products is well known. In order visualize the ‘in situ’ distribution of low molecular weight glutenins, high molecular weight glutenins, and gliadins simultaneously in wheat doughs we needed to overcome and eliminate dough auto-fluorescence, and to develop a reliable immunostaining procedure for their simultaneous detection in wheat doughs. We are studying different auto-fluorescence quenchers used in biological fluorescent imaging and their effect on dough auto-fluorescence removal, and the effect of different fixative mediums on the adhesion of wheat flours doughs onto microscope slides. We found that the best method to remove dough auto-fluorescence is removing it as background in the microscope detection system. We also found methanol to be the best fixative medium for dough samples. In this research, we are showing the first ‘in situ’ localization of these gluten subunits simultaneously in wheat flour dough.