Antigen Array Probing
Blocking and Wash Buffer: 3%
FCS in 1X PBS; 0.05% Tween 20
Incubation Buffer: 3% FCS in 1 X PBS
1. Prepare slides for blocking
- circumscribe arrays on glass slides with hydrophobic marker (PAP pen)
- use diamond scribe or marker pen to number slides
3. Incubate arrays with diluted human or animal serum samples
- most human and murine sera are used at 1:150 dilutions in incubation buffer
- place slides in histology chamber, array-side up
- before the array surface dries, put 300 µL of diluted sera on slide within the region bordered by the hydrophobic marker
- incubate at 4 C for 1 hr
Note: For sample conservation, arrays can be proved with ~ 30 µL of diluted serum samples under cover slips
4. Wash #1
- quick rinse in wash buffer
- 2 x 15 min washes in wash buffer with rotation (~40 rpm)
5. Incubate array with secondary antibody
- place slides in histology chamber, array-side up
- put 300 µL of diluted 2 Ab onto each slide
- (label is photosensitive) wrap histology chamber in aluminum foil
- incubate at 4 C for 45 minutes
6. Wash #2
- quick rinse in wash buffer
- 2 x 30 min washes in wash buffer on rotator (~40 rpm)
- quick rinse in 1X PBS
- 2 x 20 min washes in 1X PBS on rotator
- 2 x 15 sec wash in DDI H2O on rotator
7. Dry slides by centrifugation
- spin for 8 - 10 min at 25 C
- put slides in opaque slide box for transport and storage (at room temperature)
Arrays should be scanned within several days.
8. Scanning slides
- use Axon Microarray Scanner and Axon GenePix 5.0 software
