Alexa fluor 647 conjugated goat anti human igm heavy chain

Serum samples from respective cases and controls were diluted 1:1 in glycerol and stored at −20 °C until analyzed. The 125,000 random 12-mer peptide microarrays were manufactured according to the methods of Leguti et al. [21 (link)] and blocked in 0.5% BSA (Sigma, St. Louis, MO) and 1× PBS, pH 7.2. Samples were diluted to 1:1000 in 1× PBS, 0.5% BSA, and 0.05% Tween 20 pH 7.2 and exposed to the microarrays for 1 h at 37 °C with gentle agitation. After 1 h, the arrays were washed in distilled water 3× and incubated with 4 nM of Alexa Fluor 555 conjugated goat anti-human IgG (H & L) and 5 nM of Alexa Fluor 647 conjugated goat anti-human IgM heavy chain (Thermo Fisher) for 1 h at room temperature, then washed 3× in distilled water and 1× in 90% isopropyl alcohol, and dried in a centrifuge. Slides were scanned on an Innopsys 1100 scanner at 0.5-um resolution, and TIFF images were aligned in GenePix 6.0.