Gallyas silver staining was performed with a modified protocol described by Nadler et al.22. The PFA-fixed thin-layer 3D cultures were washed 3 times with deionized distilled water (DDW) for 5 min and then incubated two times with a pretreating solution containing 4.5% (w/v) NaOH and 0.6% (w/v) ammonium nitrate. The cultures were then incubated with an impregnation solution containing 5.4% (w/v) NaOH, 6.4% (w/v) ammonium nitrate and 0.3% (w/v) silver nitrate for 10 minutes followed by washings with a washing solution containing 1 ml of 0.0012% (w/v) ammonium nitrate, 0.5% (w/v) sodium carbonate and 28.5% (v/v) ethanol for 3 times with 5-min interval. The deposition of silver particles was detected by incubating with a developer solution with 0.012% (w/v) ammonium nitrate, 0.05% (w/v) citric acid, 0.56% (w/v) formalin and 9.5% (v/v) ethanol for 1–5 minutes. The reaction was stopped by adding 0.5% acetic acid.
