Thursday, May 30, 2019
Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) is a non-cleavable and membrane permeable crosslinker. It contains an amine- opposeive N -hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bondsThe staining procedure starts by placing the slides in a slide rack, immersed in a staining dish containing phosphate buffer solution and incubated for 5 min. PBS was removed from slides by tipping the slides and allowing the PBS solution to drip out. Residual PBS around the samples was also removed by gently entrancing the solution with Kim wipes without contaminating and damaging the samples. Diluted antibody solution (1/10 v/v in PBS) were directly inoculated to the regions encircled with wax pen and incubated for one moment without letting the sections dry. The slides were washed in PBS solut ion for 10 min. This step was triplicated. One drop of prolong) media as an antifading agent was applied to each section and cover with a coverslip to preserve the QDs from photobleaching during fluorescence microscopy experiments. The edges of the coverslips were sealed with nail polish to prevent drying. Slides were placed in a dark mode and we waited until the nail polish dries at room temperature for 12 hours. They were kept for another 12 hours at 4 C in a refrigerator.A confocal laser scanning microscope (Zeiss LSM 710, Carl Zeiss Micro visualise GmbH, Germany) was used to foresee dough sample microstructure. Starch granules were identified by simple polarized light .The excitation wavelengths of the QDs were 405 nm for the reflection and 615 nm for the fluoresce... ...mages of are illustrated in figure for.The results showed that antibody-quantum dots conjugates successfully diffused into the 3D matrix and were bound to gliadins. Distribution and location of gliad in at different focal planes in each section were found to show correspondent patterns for a given mixing time. Gliadins were evenly dispersed in dough sections and typically localized in the center of the sections. This supports the observation and hypothesis that the mobility of gliadin due to its cut back viscosity enables gliadin to diffuse to the inner sections of the dough along with all other parts of the sample. Intensities of gliadins at top and bottom stacks were relatively low compared to ones situated at center. It might be because of optical sectioning of starch molecules found at top and bottom surfaces of sections play a dominant part in the imaging process.