Ep1580y

CRC tissues were incubated with PBS/0.2% Triton X-100/10% dimethyl sulfoxide /6% donkey serum at 37 °C for 2 days to prevent nonspecific antigen-antibody binding and then washed with PBS/ 0.2% Tween-20, each containing 10 mg/mL heparin at 37 °C for 1 hour. Heparin was added to reduce background labeling.^{26 (link)} Several primary antibodies, including antibodies against cytokeratin 19 (EP1580Y, rabbit monoclonal; 1:200, Abcam), desmin (goat polyclonal; 1:100, LifeSpan Biosciences), CD31 (JC/70A, mouse monoclonal; 1:100, Thermo Fisher Scientific), and E-cadherin (M168, mouse monoclonal; 1:150, Abcam) were used. Up to 3 primary antibodies were simultaneously applied. The antigen retrieval step was not included. After labeling with 2 or 3 primary antibodies, tissue samples were washed 5 times for 1 hour each with PBS/0.2% Tween-20 and 10 mg/mL heparin solution. The dense desmoplastic stroma in CRCs may represent a physical barrier to antibody penetration. Therefore, the following methods were used to increase antibody penetration: (1) gradual increase in antibody concentration, (2) application of centrifugal force, (3) application of smaller molecular pepsin-digested secondary antibody fragments, and (4) sonication. For example, for cytokeratin 19, the antibody concentration was gradually increased over 4 days from an initial dilution of 1:800 to a final dilution of 1:200.