![Figure 1. Analysis of LOH in mast cells in VHL disease hemangioblastomas. Low-power (A; 10× objective) and high-power (B; 40× objective) magnification of a hemangioblastoma stained for mast cell tryptase. Note the lack of any pattern or organization of mast cells in the tumor. Vessels are indicated by arrows (A). No perivascular cuffing can be observed. Note the tendency of mast cells to occur in clusters (B). Inset shows localization of mast cell tryptase (green) and KIT receptor (red) in the same cell. The scale bar indicates 10 μm. Mast or stromal cells, identified by tryptase (C) or inhibin-A (E) immunostaining, respectively, were microdissected from frozen sections of the hemangioblastomas. Sections stained for mast cell tryptase (C-D) or for inhibin-A (E-F) are shown before (C,E) and after (D,F) microdissection. Arrows (C-D) indicate the same location in the section before and after microdissection. Peripheral blood lymphocytes were acquired from the same patients. LOH analysis was performed as described previously6,10 with minor modifications. PCR amplification products, generated using primers (D3S1038) from a microsatellite region associated with the VHL gene, are shown for a thoracic (G) and cerebellar (H) hemangioblastoma. Note the presence of allelic imbalance in the stromal (S) and mast cell (M) samples compared with the normal lymphocytes (L). Film exposures were selected to allow comparison of similar band intensities. For immunohistochemistry, primary antibodies (anti–mast cell tryptase [Thermo Fisher] and anti–inhibin-A [AbD Serotec]) were detected with MACH4 HRP-Polymer (Biocare Medical) and DAB (Vector Laboratories). Images were obtained with a Leica DMLB microscope using a spot-imaging camera and software. For the double immunofluorescence studies, anti-KIT (Sigma-Aldrich) and anti-tryptase antibodies were detected with rhodamine- and FITC-conjugated donkey secondary antibodies (Jackson ImmunoResearch). Images were obtained using Zeiss Axiophot2 microscope, Nikon Imaging Mono 12BIT camera, and Nikon NIS Elements BR software.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/5/10.1182_blood-2012-08-452151/4/zh89991200420001.jpeg?Expires=1724222473&Signature=jRfCTOIJ1nUHDyyR5S0HhJe-LuzUtxEwkmsw6EC34usyRWSEB85qrzyVr3dU2YoYvEhjCct6~16LzWqCD5hukbaFtc4eIOiuGo8RxFZgKmEfY84wb8UBTs-IXpUa7745z3m4FFSAO32U3~-ie9GDpN8edD1aT9qTEaMg~Il~~-usRJVpXo1dRV1nUVb-cPFwNXJQoxkiORR5~eEcFSUhWXUfHDcGrsQoyFQxKYE1wYYEmsBbQeW1GO4QKVKH~1ZD9ZSL6k2vReMvf01ggSSijzEluuMVAPGnV4h2ZYYlK5iePJn9LlEK9TGBuUsuB9La1nIqFhRU861UzlY8LEF89w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Analysis of LOH in mast cells in VHL disease hemangioblastomas. Low-power (A; 10× objective) and high-power (B; 40× objective) magnification of a hemangioblastoma stained for mast cell tryptase. Note the lack of any pattern or organization of mast cells in the tumor. Vessels are indicated by arrows (A). No perivascular cuffing can be observed. Note the tendency of mast cells to occur in clusters (B). Inset shows localization of mast cell tryptase (green) and KIT receptor (red) in the same cell. The scale bar indicates 10 μm. Mast or stromal cells, identified by tryptase (C) or inhibin-A (E) immunostaining, respectively, were microdissected from frozen sections of the hemangioblastomas. Sections stained for mast cell tryptase (C-D) or for inhibin-A (E-F) are shown before (C,E) and after (D,F) microdissection. Arrows (C-D) indicate the same location in the section before and after microdissection. Peripheral blood lymphocytes were acquired from the same patients. LOH analysis was performed as described previously6,10 with minor modifications. PCR amplification products, generated using primers (D3S1038) from a microsatellite region associated with the VHL gene, are shown for a thoracic (G) and cerebellar (H) hemangioblastoma. Note the presence of allelic imbalance in the stromal (S) and mast cell (M) samples compared with the normal lymphocytes (L). Film exposures were selected to allow comparison of similar band intensities. For immunohistochemistry, primary antibodies (anti–mast cell tryptase [Thermo Fisher] and anti–inhibin-A [AbD Serotec]) were detected with MACH4 HRP-Polymer (Biocare Medical) and DAB (Vector Laboratories). Images were obtained with a Leica DMLB microscope using a spot-imaging camera and software. For the double immunofluorescence studies, anti-KIT (Sigma-Aldrich) and anti-tryptase antibodies were detected with rhodamine- and FITC-conjugated donkey secondary antibodies (Jackson ImmunoResearch). Images were obtained using Zeiss Axiophot2 microscope, Nikon Imaging Mono 12BIT camera, and Nikon NIS Elements BR software.
