Preparation of tissue microarrays and immunohistochemical staining were performed as previously described [24 (link)]. The intensity of COMP was evaluated independently by two researchers in a blinded fashion using scores: 0 for negative staining, 1 for low expression, 2 for moderate expression and 3 for high expression. Staining in cancer cells was evaluated separately from the stroma.
To evaluate metastases into the lungs of mice, the first fifteen 5 μm cuts were discarded. The next tissue cut was mounted on Superfrost Plus Adhesion Microscope slides (Epredia). The antigen retrieval was performed with the heat-induced epitope retrieval method using 10 mM citrate buffer (pH = 6). Human metastatic cells were stained with an anti-pan-cytokeratin antibody (Sigma) followed by a Signalstain Boost IHC detection reagent (Cell Signaling Technology). ImmPACT DAB (Vector Laboratories) was then used for the detection of positive cells. The final mounted samples were scanned with the Aperio Scanner system (Leica) at 40X, and the number of positive cells was evaluated by QuPath software [25 (link)].
To evaluate metastases into the lungs of mice, the first fifteen 5 μm cuts were discarded. The next tissue cut was mounted on Superfrost Plus Adhesion Microscope slides (Epredia). The antigen retrieval was performed with the heat-induced epitope retrieval method using 10 mM citrate buffer (pH = 6). Human metastatic cells were stained with an anti-pan-cytokeratin antibody (Sigma) followed by a Signalstain Boost IHC detection reagent (Cell Signaling Technology). ImmPACT DAB (Vector Laboratories) was then used for the detection of positive cells. The final mounted samples were scanned with the Aperio Scanner system (Leica) at 40X, and the number of positive cells was evaluated by QuPath software [25 (link)].
Free full text: Click here
