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The proteins were then visualized by a developing solution composed by 5.7 × 10−4 M citric acid and 0.1% formaldehyde.
Addition of a HRP-conjugated secondary antibody, followed by a developing solution and a stop solution, provides a colorimetric readout which was quantified by measurement of absorbance at 450 nm with a reference wavelength of 655 nm.
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Immunodetection was performed by the addition of a developing solution.
The binding of the antibodies was demonstrated by the addition of a developing solution containing 66 mM NaHPO4, 33 mM citric acid, 0.015% H2O2, and 1 mg/mL O-phenylenediamine dichloride (OPD) compound (Sigma) for 20 min at room temperature.
The peroxidase-positive bands were detected by immersing the blots in a developing solution 4CN substrate (KPL) for 5 min. The enzyme reaction was terminated by washing the blots in 0.1 M H2SO4.
Reaction products were separated by Thin Layer Chromatography (TLC) in a developing solution made of 0.3 M LiCl, 1 M formic acid.
Stop the revelation by replacing the developing solution by 5% acetic acid.
Then, anti-M13 HRP was introduced to detect phage binding followed by ABTS developing solution.
After 20 min of staining with staining solution and two washes, protein bands were visualized by adding developing solution.
The reactions were stopped by replacing the developing solution with water.
The 40 μm sections were stained by immersing them in the developing solution.
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