Publication protocol
5 mm-thick sections were cut using semi-motorized rotary microtome (Leica MR 2145); they were then dewaxed and rehydrated through a graded ethanol series using routine protocols. Sections were then washed with distilled water and PBS for 10 min, then treated with 2% trypsin (Sigma-Aldrich) in 50 mM Tris buffer (pH 7.5), at 37°C for 15 min. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H2 O2 for 15 min to inhibit endogenous peroxidase activity. Next, the sections were incubated with primary antibodies directed against DLL1 (1:100; bs-7435R Bioss, Beijing, China), Notch1 (1:1000; bs-1196R Bioss), Jagged1 (1:100; bs-1448R, Bioss), VEGF (1:100; sc-7269, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) all for 24 h at 4°C in a humid chamber. Sections were then incubated with biotinylated secondary antibody and then with streptavidin conjugated to horseradish peroxidase (both from Zymed Histostain-Plus Peroxidase kit, 85-9043, Zymed Laboratories, San Francisco, CA, USA) prepared according to manufacturer’s instruction for 30 min each. Finally, sections were incubated with 3’,3’-diaminobenzidine (DAB) (Dead End Colorimetric TUNEL system, Promega, Madison, WI, USA) prepared according to manufacturer’s instruction, for 5 min to reveal immune labeling. All dilutions and thorough washes between stages were performed with PBS. Sections were counterstained with Mayer’s hematoxylin (Zymed Laboratories). After washing with tap water, sections were dehydrated through a graded ethanol series, cleared in xylene and mounted with Entellan (Merck, Darmstadt, Germany). Microphotographs were obtained using Olympus C-5050 digital camera mounted on an Olympus BX51 microscope. The number of positive cells was assessed systematically by scoring at least 100 cells per 10 view-fields of tissue sections at ×20 magnification independently by three histologists.
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