iRIf slide preparation

complete protocol for functionalization of iRIf slide with APTES/PDITC and Ni-NTA

duration: 2 days

select iRIf slide (clean used slides according to iRIf slide cleaning protocol)
check orientation of Tantaloxide layer → under sunlight a violet band appears on the right side
Wash with: acetone, isopropanol, aqua dest.
Dry with wafergun
Activate in plasma generator: gas flow 80 L/h, time 5 min

Prepare APTES-solution: for 5 slides:
- 18.8 mL acetone
- 1.0 mL dd aqua
- 0.2 mL APTES (always close with Parafilm)
Incubate slides for 30 min in slideholder filled with APTES-solution
Wash 2 x 5 min in slideholder with acetone
Dry with wafergun
Put slides in new slideholder and bake them in oven at 110 °C for 45 min

Prepare PDITC-solution: for 5 slides:
- pyridin 2.0 mL
- DMF 18.0 mL
- PDITC 40 mg
Cool down slides in slidebox with wafergun (go close with gun)
Incubate slides in slideholder for 2 h filled with PDITC-solution at RT
Discard PDITC-solution
Fill with ethanol and discard ethanol
Wash 2 x 5 min in slideholder with ethanol
Wash for 5min with acetone in slideholder
Discard acetone
Dry with wafergun
Dry for 15 min in desiccator (Exsiccator)

prepare 80 µL 457 mM AB-NTA, 1 M NaHCO₃ solution per slide sandwich
- use 0.06 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.042 g NaHCO₃ in 500 µL aqua dest
- use 0.096 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.067 g NaHCO₃ in 800 µL aqua dest
- use 0.108 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.076 g NaHCO₃ in 900 µL aqua dest
put 80 µL on one slide and put the other one upon → Make sure the upper sides were laid together
incubate o/n
prepare blocking solutions:
- blocking solution for GOPTS: 10mg/ml BSA + 0.5% ethanolamine in PBS
- blocking solution for APTES/PDITC:5mg/ml BSA + 5% ethanolamine in PBS
transfer slides into blocking solution for 30 min-60 min
2x wash slides for 10 min in PBS (shake it)
prepare 20 mL 1 % (w/v) NiSO₄ solution per 5 slides (= 0.2 g/20 mL)
incubate slides in slide holder with nickel sulfate for 1 h
2x wash slides for 10 min in PBS (shake it)
prepare 1/10 PBS-dilution in aqua dest and check for pH (should be 7-8)
wash slides in diluted PBS-solution
Dry slides with wafer gun

spot using 11-spot mask
(use 3 µl of bBSA (200 µg/ml in 1mg/ml BSA) as control spot)
incubate o/n @ 4°C
remove protein solution with pipette
incubate twice 5 min with 10 mg/mL BSA solution in the wells
(flush slide with PBS)transfer slide into PBS solution and remove mask
block slides with BSA (10 mg/ml) for 30 min
wash 10 min in PBS
wash 10 min in PBS (with 20 mM Imidazol, 27.2 mg in 20 mL PBS)
wash 10 min in 1/10 diluted PBS
dry with wafer gun
measure in iRIf setup
if Strep-Cy5 solution was flushed over slide during iRIf measurement also measure Cy5 fluorescence in microarray scanner afterwards
store iRIf slides in 0.1 % SDS o/n

Team:Freiburg/Protocols/SlidePrep