Epoxy-functionalized
glass slides (Super
epoxy 2, Arrayit Corporation) were coated with 100 μg/mL ovalbumin
in PBS overnight at 4 °C and then rinsed with PBS and blocked
with 1 mg/mL BSA/PBS for 30 min. After incubation of the cell-loaded
array, the ovalbumin-coated glass slide was then placed onto the loaded
array for printing. The microarray and glass slide were held together
by compression in a hybridization chamber (Agilent Technologies, G2534A)
and incubated for 2 h at 37 °C with 5% CO2 as described
previously.6 (link) The glass slide was then separated
from the array and placed in PBS. After microengraving, slides were
incubated for 30 min with blocking buffer (PBS, 3% w/v milk powder
and 0.05% (v/v) Tween-20), washed with PBST (PBS + 0.05% v/v Tween-20),
and then incubated with fluorescent antibodies (Alexa Fluor 488 goat
anti-mouse IgG) at 2 μg/mL for 45 min at 25 °C. The slides
were washed with PBST and PBS, rinsed briefly with water, and dried
with a N2 stream. Slides were scanned using a Genepix 4200AL
microarray scanner (Molecular Devices). The median fluorescence intensity
of each element was analyzed using Genepix Pro. Data extracted from
both on-chip cytometry and printed proteins were matched in Microsoft
Excel using unique identifiers assigned to each well within the array.
The dataset was filtered to include wells containing only single cells.
glass slides (Super
epoxy 2, Arrayit Corporation) were coated with 100 μg/mL ovalbumin
in PBS overnight at 4 °C and then rinsed with PBS and blocked
with 1 mg/mL BSA/PBS for 30 min. After incubation of the cell-loaded
array, the ovalbumin-coated glass slide was then placed onto the loaded
array for printing. The microarray and glass slide were held together
by compression in a hybridization chamber (Agilent Technologies, G2534A)
and incubated for 2 h at 37 °C with 5% CO2 as described
previously.6 (link) The glass slide was then separated
from the array and placed in PBS. After microengraving, slides were
incubated for 30 min with blocking buffer (PBS, 3% w/v milk powder
and 0.05% (v/v) Tween-20), washed with PBST (PBS + 0.05% v/v Tween-20),
and then incubated with fluorescent antibodies (Alexa Fluor 488 goat
anti-mouse IgG) at 2 μg/mL for 45 min at 25 °C. The slides
were washed with PBST and PBS, rinsed briefly with water, and dried
with a N2 stream. Slides were scanned using a Genepix 4200AL
microarray scanner (Molecular Devices). The median fluorescence intensity
of each element was analyzed using Genepix Pro. Data extracted from
both on-chip cytometry and printed proteins were matched in Microsoft
Excel using unique identifiers assigned to each well within the array.
The dataset was filtered to include wells containing only single cells.
