Dab plus kit

Slices of paraffin-embedded tissue (4 μm thick) from 5 pairs of matched samples adjacent-mucosa tumor tissue were used. For antigen retrieval, the slides were boiled after deparaffinization in a pressure cooker for 10 minutes in citrated buffer (8.2 mM tri-sodium citrate and 1.98 mM citric acid, pH6) for Robo2 detection and in EDTA buffer (1 mM EDTA, 0.05% Tween-20, pH8) for Slit2 detection. Endogenous peroxidase was blocked with 3% H₂O₂ during 20 minutes. After blocking during 30 minutes with 1/5 dilution of goat serum, primary antibodies were incubated overnight at 4°C. Primary antibodies were rabbit polyclonal against Slit2 (Abcam, ab111128) and rabbit polyclonal against Robo2 (Prestige Antibodies, HPA013371), diluted both 1:100 in antibody diluent (Dako, Copenhagen, Denmark). Reaction was visualized using EnVision anti-rabbit antibody system, and developed using DAB-Plus Kit (Dako). Slides were counterstained with Harry’s modified haematoxylin. As negative control we used EnVision anti-rabbit antibody system and displayed no reactivity against any antigen.

For histologic analyses, a tumor fragment was fixed in formalin for 24 h, embedded in paraffin and stained with H&E. The histology of tumor PDOX was compared with the histology of extramedullary cutaneous lesion from which the PDOX was derived by a haematopathologist. For IHQ analysis, 3-µm slices of paraffin-embedded tissues were used. Primary antibodies used were monoclonal antibodies for Ki67 (RM-9106-51, Sigma-Aldrich) and CD38 (NB-22-8022, Neobiotech), and were used at a 1:200 dilution. Retrieval was performed with citrate buffer (pH 6.0) using a pressure cooker. Reactions were visualized using the EnVision anti-mouse antibody system, and developed using the DAB-Plus Kit (Dako, Copenhagen, Denmark). Slides were counterstained with Harry's modified Hematoxylin.