For the construction of tissue microarrays (TMA), 1 mm tissue cylinders were extracted from formalin-fixed and paraffin-embedded tumor samples archived in the department Neuropathology after evaluation and marking of the respective hematoxylin and eosin stains. For most of the cases, enough tissue was available to retrieve two tissue probes. A conventional microarrayer was used (Beecher Instruments, Sun Prairie, WI, USA). TMAs were cut with a microtome, and 4 μm tissue slices were produced and dried at 80° for 15 min. Immunohistochemical staining was done with a Ventana BenchMark immunostainer (Ventana Medical Systems, Tucson, AZ, USA).
Pretreatment with cell conditioning solution CC1 (pH 8.5) was done for 14 (CD68), 40 (MIB1, CD3 and CD163) or 64 min (CD8) followed by primary antibody incubation at 37° (CD8 (ready to use, Roche, Basel, Switzerland) and CD163 (1:1000, ABD Serotec, Puchheim, Germany)) or 42 °C (MIB1 (1:200, DAKO, Santa Clara, CA, USA), CD3 (1:500, Thermo Fisher Scientific, Waltham, MA, USA) and CD68 (1:200, Agilent DAKO, Santa Clara, CA, USA)). Subsequently, OptiView HQ universal linker was applied for 12 min, followed by incubation with OptiView HRP Multimer for 12 min. Counterstaining was done with hematoxylin for 4 min.
As controls, the human cerebral and cerebellar cortex, as well as a sample of a colorectal carcinoma metastasis, were placed on each TMA block.
Pretreatment with cell conditioning solution CC1 (pH 8.5) was done for 14 (CD68), 40 (MIB1, CD3 and CD163) or 64 min (CD8) followed by primary antibody incubation at 37° (CD8 (ready to use, Roche, Basel, Switzerland) and CD163 (1:1000, ABD Serotec, Puchheim, Germany)) or 42 °C (MIB1 (1:200, DAKO, Santa Clara, CA, USA), CD3 (1:500, Thermo Fisher Scientific, Waltham, MA, USA) and CD68 (1:200, Agilent DAKO, Santa Clara, CA, USA)). Subsequently, OptiView HQ universal linker was applied for 12 min, followed by incubation with OptiView HRP Multimer for 12 min. Counterstaining was done with hematoxylin for 4 min.
As controls, the human cerebral and cerebellar cortex, as well as a sample of a colorectal carcinoma metastasis, were placed on each TMA block.