Deparaffinised slides for immunohistochemical staining underwent heat antigen retrieval for 20 min in pre-heated 100°C Dako EnVision™ Flex Target Retrieval Solution pH 9.0 (K8023, DakoCytomation, Carpinteria, CA, USA). Slides were cooled for 45 min and then placed in a Tris-buffered saline (TBS) bath for 10 min.
Immunohistochemical staining was performed using the Dako Autostainer Model S3400 (DakoCytomation). A 3% hydrogen peroxide solution (DakoCytomation) was applied for 10 min followed by 30-min incubations with the primary antibodies monoclonal mouse anti-human CD31, endothelial cell clone, JC70A and M0823 (1:400) and monoclonal mouse anti-human D2-40, clone D2-40 and M3619 (1:50), and then incubated for 30 min with the detection system EnVision + Dual Link System-HRP K4061 (DakoCytomation). The reaction was visualised with a 10-min incubation of liquid DAB + Substrate Chromogen System K3468 (DakoCytomation). Slides were counterstained with Mayer’s haematoxylin and cover-slipped.
Immunohistochemical staining was performed using the Dako Autostainer Model S3400 (DakoCytomation). A 3% hydrogen peroxide solution (DakoCytomation) was applied for 10 min followed by 30-min incubations with the primary antibodies monoclonal mouse anti-human CD31, endothelial cell clone, JC70A and M0823 (1:400) and monoclonal mouse anti-human D2-40, clone D2-40 and M3619 (1:50), and then incubated for 30 min with the detection system EnVision + Dual Link System-HRP K4061 (DakoCytomation). The reaction was visualised with a 10-min incubation of liquid DAB + Substrate Chromogen System K3468 (DakoCytomation). Slides were counterstained with Mayer’s haematoxylin and cover-slipped.