Array pro analyzer ver 6

The plates were thawed at room temperature, the blocking buffer was discarded, and was treated with serum diluted by 3:1000 in the reaction buffer containing 1x Synthetic block (Invitrogen), phosphate-buffered saline, and 0.1% Tween 20. After the reaction for 1 hour, the plates were washed twice with TBST (#9997; Cell Signaling Technology). Next, the plates were treated with goat anti-human IgG (H+L) Alexa Flour^® 647 conjugate (A21445; Thermo Fisher Scientific) diluted in the reaction buffer for 1 hour, washed twice with TBST, washed again under running reverse osmosis water, and air-dried. Finally, the plates were scanned at a 20-µm resolution using a Typhoon FRA 9500 (GE Healthcare) fluorescent imager. The scanned images were saved as 16-bit tiff files. Array-Pro Analyzer ver. 6.3.1 (Media Cybernetics) was used to record the median by drawing an equal-sized circle around the spots. The negative controls were prepared using distilled water instead of mRNA during protein preparation. The positive controls were prepared using mRNA coding IgG for protein synthesis.

The blocking buffer on the plates was thawed at room temperature and discarded. The plates were treated with serum diluted 3:1000 in PBS containing 0.1% skim milk, 1x Synthetic Block (PA017, Invitrogen, Waltham, MA, USA), and 0.1% Tween 20. The amount of serum needed per test was 300 µL. After the reaction at room temperature for 1 h, the plates were washed 3 times with TBST and incubated with goat anti-human IgG (H+L) Alexa Flour^® 647 conjugate (A-21445, Invitrogen) diluted 1:1000 in PBS containing 1x Synthetic Block and 0.1% Tween 20 at room temperature for 1 h. Then the plates were washed twice with TBST, washed again with running reverse osmosis water, and air-dried. Finally, the plates were scanned at a 100-µm resolution using a GenePix 4000B Microarray Scanner (Molecular Devices, San Jose, CA, USA). The scanned images were saved as 16-bit tiff files. Array-Pro Analyzer ver. 6.3.1 (Media Cybernetics, Rockville, MA, USA) was used to determine the median value within each spot for signal quantification. The negative control spots were prepared using distilled water (10977015, Invitrogen) instead of mRNA during protein preparation. The positive control spots were prepared using mRNA encoding human IgG for protein synthesis.