| ID |
2 |
| Publication |
10.1038/srep43560
|
| date of publication |
2017/3/6
LM-GlycomeAtlas
|
| Species |
Mouse |
| Supplier |
Leica |
| Instrument |
LMD7000 |
| Observation Type |
Bright-field |
| Detail Description |
Tissue dissection were performed using LMD with a Leica LMD6000 (Leica Microsystems, Wetzlar, Germany). Each microdissected tissue fragment (~0.9 mm^2) was collected into a 0.5-mL tube and the tubes were centrifuged at 20,000 x g for 1 min at 4∘C, followed by adding 200 µL of 10 mM citrate buffer (pH 6.0). Antigen retrieval procedure was performed by incubating the tubes containing the tissue fragments and buffer at 95∘C for 1 h. After the addition of 4 µL of a 50% slurry of microcrystalline cellulose into the tube and a wash with phosphate-buffered saline (PBS) by centrifugation, the tissue pellets were solubilized with 20 µL of PBS containing 0.5% Nonidet P-40 and sonicated three times for 10 sec each. Subsequently, the tissue suspension was incubated on ice for 1 h and centrifuged at 20,000 x g for 1 min at 4∘C. The obtained supernatants (20 µL) were collected as crude protein extracts. |
| Lectin Array Chip |
download (Excel) |
| Sample preparation |
The obtained supernatants (20 µL) were fluorescence-labeled with 10 µg of Cy3-succinimidyl ester (GE Healthcare, Buckinghamshire, UK) at RT for 1 h in the dark. The sample solution was adjusted to 100 µL with probing buffer (500 mM glycine, 1 mM CaCl2, and 1 mM MnCl2 in Tris-buffered saline containing 1.0% Triton X-100) and incubated at RT for 2 h to block the excess fluorescent reagent. |
| Supplier of instrument |
GlycoTechnica |
| Instrument |
GlycoStation Reader1200 |
| Data acquisition |
Each 60 µL aliquot of Cy3-labeled glycoprotein was applied to one well on a lectin microarray chip (LecChip, GlycoTechnica, Yokohama, Japan) and incubated 16 h at 20∘C. Fluorescence signals were measured using a chip scanner GlycoStation Reader 1200 (GlycoTechnica), and the data were analyzed with Array-Pro Analyzer software (Version 4.5, Media Cybernetics, Bethesda, MD, USA). |
| Image analysis software |
Array-Pro Analyzer software (Version 4.5, Media Cybernetics, Bethesda, MD, USA) |
| Data processing and statistical analysis |
The net intensity was calculated by subtracting the background from the signal intensity. The scanning data under appropriate gain conditions, which provided the net intensities of all positive spots < 40,000. The relative intensity of each lectin was normalized using the mean of signal intensities of all lectins. |
