Anti pan cytokeratin

ScSCREENCELL CYTO kits were used for immunostaining of cancer cells isolated from the blood. For immunostaining, the protocol provided by SCREENCELL was followed. Briefly, cytofilters used for filtration were washed with 1× Tris-buffered saline (TBS) for 10 min. For antigen retrieval, samples were treated with Target Retrieval Solution (S1700; Dako) at 95°C for 20 min. The cytofilters were allowed to cool to room temperature before rinsing with 1× TBS, followed by a 5 min incubation in permeabilizing buffer (1× TBS, 0.2% Triton X-100). Next, the samples were incubated in 3% BSA at room temperature for 30 min. Cytofilters were subsequently incubated with the following primary antibodies in parallel in 3% BSA at 4°C overnight: anti-Pan Cytokeratin (MA5-17687; Dako) at 1:50 and anti-EpCAM (Ab71916; Abcam) at 1:100. After rinsing with Wash Buffer (1× TBS, 0.05% Tween 20), the samples were stained with the following secondary antibodies in parallel at 1:1,000 in 3% BSA for 1 hr at room temperature: goat anti-mouse Alexa 488 and goat anti-rat Alexa 594 (A11001, A11007; Life Technologies). Finally, cytofilters were rinsed with Wash Buffer and protected with ProLong Diamond Mountant containing DAPI nuclear stain (P36962; Life Technologies). Images were acquired using a Leica SP8 STED at the University of Iowa Central Microscopy Research Facility.