| ID |
4 |
| Publication |
10.1016/j.reth.2022.12.009
|
| date of publication |
2022/12/22
LM-GlycomeAtlas
|
| Species |
Mouse |
| Supplier |
Leica |
| Instrument |
LMD7000 |
| Observation Type |
Bright-field |
| Detail Description |
Tissue sections were cut to 0.5mm^2 in the same region using LMD7000 (Leica Microsystems, Wetzlar, Germany) and collected into microtubes. For proteins extraction, 200 mL of 10 mM citrate buffer
(pH 6.0) was added to each tissue fragment and incubated at 95∘C for 1 h. A 50% slurry of 20 mm cellulose (SigmaeAldrich, Co., St. Louis, MO, USA) was added to the tube and thenwashed twice with PBS. After centrifuging at 20,000 x g for 1 min at 4∘C, the slurry was solubilized with 20 mL of PBS containing 0.5% Nonidet P-40 (Nacalai Tesque, Inc., Kyoto, Japan) and subjected to three cycles of sonication (15 sec each). Thereafter, the sonicated solution was incubated on ice for 1 h and collected by centrifugation at 20,000 x g for 1 min at 4∘C. The supernatant was resuspended in 20 mL PBS. |
| Lectin Array Chip |
download (Excel) |
| Sample preparation |
The supernatant was resuspended in 20 mL PBS and labeled with 10 µg of Cy3 mono-reactive dye (GE Healthcare, Buckinghamshire, UK) at 25∘C for 1 h in the dark. The volume of the reaction solution was adjusted to 100 mL with probing buffer (Trisbuffered saline containing 1% Triton X-100, 1 mM CaCl2, and 1 mM MnCl2; pH 7.4) and incubated at 25∘C for 2 h. |
| Supplier of instrument |
GlycoTechnica |
| Instrument |
GlycoStation Reader1200 |
| Data acquisition |
The Cy3-labeled solution (60 µL) was applied to a LecChip (GlycoTechnica, Yokohama, Japan) and incubated at 25 C for approximately 17 h. Thereafter, the LecChip was washed three times with probing buffer and then analyzed on an evanescent-field fluorescence scanner (GlycoStation Reader 1200, GlycoTechnica) using the GlycoStation Tools Signal Capture 1.0 and GlycoStation Tools Pro 1.0 software applications. |
| Image analysis software |
GlycoStation Tools Pro Suite 1.0 |
| Data processing and statistical analysis |
Scanning data were selected under appropriate gain conditions, which satisfied fewer than 63,000 net intensities of all signals, and averaged data were analyzed for glycan characterization. For accurate comparative analysis, the data were used with average-normalization. |
