| ID |
1 |
| Publication |
10.3390/molecules24162962
|
| date of publication |
2019/8/15
LM-GlycomeAtlas
|
| Species |
Mouse |
| Supplier |
Leica |
| Instrument |
LMD7000 |
| Observation Type |
Bright-field |
| Detail Description |
Tissue dissection using an LMD7000 LMD system (Leica Microsystems) and subsequent protein extraction were performed, tissue fragments (total area of 0.13-0.91 mm^2) were dissected from hematoxylin-stained sections and collected in a tube. After centrifugation at 20,000 g for 1 min at 4∘C, the fragments were heat-denatured in 200 µL of 10 mM citrate buffer, pH 6.0 for 1 h at 95∘C. After centrifugation at 20,000 g for 1 min at 4∘C, 4 µL of a 50% (w/v) slurry of microcrystalline cellulose in Dulbecco's PBS (D-PBS) was added. After discarding 190 µL of the supernatant and adding 190 µL of D-PBS, the fragments were subjected to protein extraction using 20 µL of D-PBS containing 0.5% Nonidet P-40 by sonication. After incubation for 1 h on ice and centrifugation at 20,000 x g for 1 min at 4∘C, the supernatants were collected as protein extracts. |
| Lectin Array Chip |
download (Excel) |
| Sample preparation |
20 µL of protein extracts were fluorescently labeled with Cy3-succinimidyl ester (10 µg protein equivalent; GE Healthcare, Buckinghamshire, UK) for 1 h at RT in the dark. After adjusting the volume to 100 µL with probing buffer (25 mM Tris-HCl, pH 7.5, containing 137 mM NaCl, 2.7 mM KCl, 500 mM glycine, 1 mM CaCl2, 1 mM MnCl2, and 1% Triton X-100), the sample solutions were incubated for 2 h at RT to quench the excess reagent. After appropriate dilution with the probing buffer when necessary, the sample solutions were added to a well (60 µL/well) on a lectin array chip (LecChip ver.1.0; GlycoTechnica, Yokohama, Japan) containing triplicate spots of 45 lectins. After incubation overnight at 20∘C and washing three times with the probing buffer, the array chip was scanned using an evanescent-field fluorescence scanner (GlycoStation Reader 1200; GlycoTechnica). |
| Supplier of instrument |
GlycoTechnica |
| Instrument |
GlycoStation Reader1200 |
| Data acquisition |
After incubation overnight at 20∘C and washing three times with the probing buffer, the array chip was scanned using an evanescent-field fluorescence scanner (GlycoStation Reader 1200; GlycoTechnica). All data were analyzed using the GlycoStation Tools Pro Suite 1.5 software (GlycoTechnica). |
| Image analysis software |
GlycoStation Tools Pro Suite 1.5 |
| Data processing and statistical analysis |
The net intensity was calculated by subtracting the background from the signal intensity. The data under appropriate gain conditions providing net intensities below 40,000 for all spots were used to obtain glycomic profiles, which are presented as the relative signal intensities of the 45 lectins normalized with the mean value of all lectin signals. The limit of detection calculated as the mean +3 SD for the NPA signal intensity of the negative control was 0.1 mm^2 for the analysis of the lobules of the pancreas. |
