Panoramic 250 system

Brain tissue biopsies were collected from four human fetuses electively aborted because of HCMV infection and from two controls aborted for Di George syndrome with associated cardiopathy (control case 1200496) or for premature rupture of membranes, anamnios, and chorioamnionitis (control case 1200094). Immunohistochemical brain analysis of controls and HCMV cases was performed on sections cut at 8 μm from paraffin blocks, using standard methods. Antigen was retrieved through two consecutive incubations (11 and 10 min) in a microwave oven for DCX or one 40-min incubation at 95 C in a heating bath for LIS1. Endogenous peroxidase activity and biotin were blocked by using the avidin-biotin and peroxidase blocking reagents (Vector Labs, Burlingame, CA, USA) for 10 min at room temperature. Primary antibodies were diluted 800-fold (DCX) or 250-fold (LIS1) in blocking buffer (3% BSA in PBS). Antibody specificity was ascertained by parallel staining experiments using either no primary antibody or a non-specific isotype control.
Staining for LIS1, DCX, and Mayer's haematoxylin counterstain was performed using an Autostainer device (Dako, Glostrup, Denmark), and resulting slides were scanned using a Panoramic 250 system (3D Histech, Budapest, Hungary) and analysed with the dedicated software (3D Histech).