Step-by-step instructions

Notes here

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Reagents required

 

Protocol

  1. Assemble required number of array slides with chambers into FAST frames. (Note: check the seals on the chambers since these tend to be distorted with repeated use of chambers).
  2. Prepare Blocking Buffer with 10% E. coli lysate (Note: you require 0.5ml per serum sample. Lyophilized E. coli lysate is reconstituted with Whatman Blocking Buffer, 1ml per vial)
  3. Dilute serum to 1/100-1/200 in 0.5ml Blocking Buffer/10% E. coli lysate, vortex briefly and incubate for 0.5-1h at room temperature. (This step is required to block anti-E. coli antibodies in human serum).
  4. Meanwhile, block arrays by adding 0.5ml Whatman Blocking Buffer (no lysate) to each. Incubate for at least 30 minutes with gentle rocking. Do not allow slides to dry out from now on.
  5. After blocking, remove Blocking Buffer from arrays (aspirate or ‘flick’ out contents down sink) and replace with 0.5ml diluted serum. Incubate in humidified box with gentle rocking at 4oC overnight.
  6. Wash 6x 5 minutes in T-TBS on platform shaker. (Note: for the first two washes, use vacuum aspirator to minimize cross-contamination between wells and rinse tip in T-TBS between arrays. The subsequent washes can be performed by the ‘flick’ technique).
  7. Add 0.5ml biotinylated secondary antibody diluted 1/200 in Blocking Buffer (no lysate). Incubate 1-2h in humidified box at room temperature on platform shaker. (Note: we use antibodies from Jackson Immuno – see “Resources” page; add equal volume of glycerol to reconstituted antibodies and store at -20oC). Do a secondary antibody and tertiary reagent without primary serum as a control for each print run.
  8. Wash as in step 7, but use the ‘flick’ technique throughout.
  9. Add 0.5ml streptavidin-fluorophore diluted 1/200 in Blocking Buffer. (Note: we use Streptavidin-SureLight®P3 from Colombia Biosciences; add equal volume of glycerol to reconstituted antibodies and store at -20oC). Incubate 30 minutes in humidified box at room temperature on platform shaker in the dark.
  10. Wash 3x 5 minutes in T-TBS, followed by 3x 5 minutes in TBS. Use the ‘flick’ technique throughout.
  11. Flick FAST frame one last time and remove slides. Dip slide in distilled water and centrifuge dry in 50ml centrifuge tube (~1200 rpm for 5 minutes). (Note: centrifugation eliminates drying marks. Process no more than 2 frames at a time; work quickly to prevent arrays drying out before they are centrifuged.)
  12. Scan slides, or store in the dark (in a slide box) preferably in a desiccator to scan later.