Ro¨hner et al. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis Figure 1 Alcian blue staining of chondrocyte after exposure to antiseptics. Chondrocytes were treated for 30 minutes with PBS, polyhexanide, or hydrogen peroxide, and were analysed by light microscopy. (A) PBS-treated control chondrocytes; (B) chondrocytes incubated with polyhexanide; (C) chondrocytes treated with hydrogen peroxide. One representative is shown from at least three independently performed experiments. cleaned sections were irrigated with 3% acetic acid for 3 minutes and were stained with 1% Alcian blue in 3% acetic acid (pH 2.5) for 30 minutes. After a short irrigation with 3% acetic acid (pH 2.5) and distilled water for three times, the slides were coverslipped with Vitro-Clud-coverslipping med- ium. With the high concentration of proteoglycans that are characteristic for extracellular cartilaginous matrix, the sections acquired an intensive blue colouring. Light microscopy analysis of sections was performed using an Axiovert 40C light micro- scope (Zeiss, Go¨ ttingen, Germany). The view ﬁelds were digitized using a digital camera (Canon EOS 500D, 15.1 Megapixels), and the photographs were analysed using ImageJ (GNU License) by manually deﬁning regions of interest in representative cell/ matrix areas and areas without matrix or cells for blank. The blue level of colour histograms (8 bit for blue, red, and green) of both regions was measured. The matrix proteoglycan concentration was assum- ed to be positively correlated with the blue level value, i.e. a higher blue value represents a higher proteoglycan concentration. Detection of cell number and morphological characteristics Chondrocytes embedded in alginate beads were incubated with polyhexanide or hydrogen peroxide, sectioned into 4-mm sections, and stained with 1% Alcian blue. PBS-treated chondrocytes were used as control. Analysis of the plates was performed by using Axiovert 40C light microscope (Zeiss). Cell number was detected by counting cells per view ﬁeld (lens: 1060.25, ocular: 10618). Statistical analysis Statistical tests were performed using Graph Pad Prism Software (Graph Pad Software, San Diego, CA, USA). Data were expressed as mean¡standard deviation of the mean (SEM). Statistical differences were veriﬁed using a non-parametric Wilcoxon matched-pairs test as indicated in the legends. A P- value of ,0.05 was considered signiﬁcant. Results Histological analysis by Alcian blue staining showed a signiﬁcantly decreased intensity of blue coloring after antiseptics treatment for 10 and 30 minutes Figure 2 Intensity of Alcian blue after treatment with polyexanide or hydrogen peroxide. Chondrocytes treated with antiseptics were compared with PBS-treated chondrocytes (control) after (A) 10 and (B) 30 minutes. n54, mean¡SEM, non-parametric Wilcoxon matched-pairs test. As compared to CTR, *P,0.05. Journal of Histotechnology 2011 VOL. 34 NO. 1 37