Tissue microarray tma workstation

Cytospin preparations were fixed with paraformaldehyde 3.7% in PBS for 10 minutes to room temperature and permeabilized with 0.2% triton X-100 (10 minutes). Anti-p27 (C-19) and anti-Myc antibodies (N-262, all antibodies were rabbit polyclonals from Santa Cruz Biotech.) were incubated overnight, and Texas Red or FITC-conjugated secondary antibody (Dako) were used to detect the presence of p27 and Myc. Samples were mounted with Vectashield (Vector) containing 4’-6-diamidino-2-phenylindole (DAPI) to stain nuclei and photographed under a fluorescence microscope. For immunohistochemistry, the CLL, sections of CLL lymph nodes were arrayed into a new paraffin block using a tissue microarray (TMA) workstation (Beecher Instruments, Silver Spring, MD). Immunohistochemical staining for Myc (Y29 rabbit polyclonal antibody from Dako) and p27 (SX53G8 monoclonal antibody fom Dako) was performed following conventional automated protocols in a Autostainer Plus device (Dako).

For GATA6 determination, optimal tissue blocks were selected by an expert pathologist on hematoxylin and eosin (H&E) slides. Representative tumor areas of each case were selected for tissue microarray (TMA) construction. Two representative cores of 1.2 mm in diameter were taken and arrayed into a receptor block using a tissue microarray (TMA) workstation (Beecher Instruments, Silver Spring, MD, USA) as previously described [16 (link)]. Four micrometer sections of the TMAs were used for immunohistochemistry (IHC) purposes. Briefly, slides were cut with a semiautomatic microtome HM 3508 (MICROM), deparaffinized, and rehydrated in water. Antigen retrieval was performed using a DAKO PT Link. Peroxidase activity was blocked with a Dako Protein block for 10 min, incubated for 30 min with primary antibodies, detected with a Dako Envision Plus kit, and counterstained with haematoxylin. All reagents were from Dako (Agilent, Santa Clara, CA, USA). GATA-6 antibody used: ref. number AF1700 (R&D Systems, MN, USA).