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Protocol
Instructions
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Coating buffer (MXPE74)
0.05 M carbonate-bicarbonate buffer (pH 9.6).
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Dissolve the reagents in the distilled water.
Store at +4°C.
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PBS (pH 7.4)
- 8 g of NaCl
- 0.2 g KCl
- 1.44 g Na2PO4
- 0.24 g KH2PO4
- H2O (distilled)
Dissolve NaCl, KCl, Na2PO4 and KH2PO4 in 800 ml of distilled water and adjust the pH to 7.4 using HCl.
Adjust the volume to 1 liter and store at room temperature.
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Washing buffer (PBS-Tween)
PBS + 0,05% Tween 20.
Store at room temperature.
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Sera and conjugate diluent buffer
PBS-Tween (washing buffer) + 1% Yeast extract.
Prepare minimum needed volume each day and use it fresh.
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Chromophore/Substrate solution (MXY70G)
Dissolve tablets of OPD (SIGMA) in the appropriate volume of buffer (e.g. one tablet of 60 mg OPD in 120 ml of phosphate-cytrate buffer, pH 5). This solution can be stored at –20°C in appropriate aliquots (6, 12, 18 or 24 ml aliquots, suited respectively for 1, 2, 3 or 4 plates) in the dark. Immediately before use add H2O2 at 0.02% final concentration (e.g. H2O2 30% 4 ?l H2O2 per 6 ml OPD).
Potential carcinogenic agent R45.
Store at 4 °C.
Phosphate-cytrate buffer (pH 5).
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Preparation
Dissolve tablets of OPD (SIGMA) in the appropriate volume of buffer (e.g. one tablet of 60 mg OPD in 120 ml of phosphate-cytrate buffer, pH 5). This solution can be stored at –20°C in appropriate aliquots (6, 12, 18 or 24 ml aliquots, suited respectively for 1, 2, 3 or 4 plates) in the dark. Immediately before use add H2O2 at 0.02% final concentration (e.g. H2O2 30% 4 ?l H2O2 per 6 ml OPD).
Potential carcinogenic agent R45.
Store at 4 °C.
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Blocking solution
Add H2SO4 96% (56 ml) to the distilled water (944 ml).
Store at room temperature.
