Antigen Microarray Site


 

Array Protocol

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

2.  Block slides at 4° C for 4 hours to overnight using a stainless steel slide rack in a histology dish

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 arrays with secondary antibody

  • place slides in histology chamber, array-side up

  • put 300 μL of diluted 2° Ab onto each slide

  • wrap histology chamber in aluminum foil (fluorochromes are photosensitive) 

  • incubate at 4° C for 30 - 60 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 15 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 in a table-top centrifuge

  • put slides in opaque slide box for transport and storage (at room temperature),  arrays should be scanned within one week

8.  Scanning slides

  • use Axon Microarray Scanner and Axon GenePix 3.0 software (or equivalent)