Differential staining for CD44 on cells isolated from fresh tumour samples using different methods.

<p>Panels A–D illustrate the procedure used to identify staining patterns. Cell smears were prepared and co-stained with DAPI (A) with a pan-keratin antibody (B) and with the CD44 “epitope 1” antibody (C). These panels show images manipulated by adjusting capture thresholds in PhotoShop to identify cells with set levels of staining above a background level. For each field, this level was held constant to generate the three images, one for each fluorochrome, containing only the stained cells. These images were then combined, slightly out of lateral register, to allow identification of cells with absent or low levels staining for CD44 or keratin (blue nuclei only), staining for CD44 only (CD44+), keratin only (K+), or both CD44 and keratin (CD44+K+). Treatment with trypsin+collagenase or collagenase alone consistently reduced the fraction of cells that was positive for CD44 (E) and for both CD44 and keratin (F). Displaying the 5 assayed tumours individually (G) demonstrates the striking variation in CD44 staining between tumours, and that this variation is reduced following enzymatic treatment. Statistical analysis was conducted using a Mann-Whitney test.