Ccd fv2t digital camera

Mice were anesthetized with a mix of Dormitor (1 mg/kg) and Nimatek (75 mg/kg). Tissue processing and IHC staining were performed as described [9, 32, 33] . Briefly, mice were perfused transcardially with PBS (pH 7.4) followed by 4% paraformaldehyde (PFA). Brains were isolated, post-fixed with 4% PFA overnight, and kept in 70% ethanol prior to paraffin embedding. Routinely, paraffin sections (7 µm) were used for immunofluorescent stainings. The following primary antibodies were used: polyclonal rabbit anti-IBA1 (1:500; Wako D19-19741), rat anti-F4/80 (1:500; Serotec, Oxford, UK). After incubation with primary antibodies overnight at room temperature HRP-labeled secondary antibodies (1:200) were applied for 1 hour, followed by fluorescent labeling with a cyanine 2 (FITC) TSA kit (Perkin Elmer Life sciences, Boston, USA). When double immunolabeling was performed, sets of primary and secondary antibodies were applied sequentially. As second fluorescent labels, cyanine 3 TSA kits (Perkin-Elmer) were used. Images were acquired with a motorized inverted IX-81 microscope connected to a CCD-FV2T digital camera (Olympus, Aartselaar, Belgium) and processed with LSM Image browser software (Zeiss, Germany).

Anesthesia of the mice and tissue processing for immunohistochemical (IHC) staining were performed as described [19] . Paraffin sections (7 µm) were used for immunofluorescent staining. The following primary antibodies were used: polyclonal rabbit anti-Iba1 (1:500; Wako D19-19741), rabbit anti-PBR (TSPO) (1:100; Abcam ab109497), rabbit anti-GFAP (1:200; Sigma G9269). After overnight incubation with primary antibodies at room temperature, HRP-labeled secondary antibodies (1:200) were applied for 1 hour, followed by fluorescent labeling with a cyanine 2 (FITC) TSA kit (Perkin Elmer Life sciences, Boston, USA). When double immunolabeling was performed, sets of primary and secondary antibodies were applied sequentially. The cyanine 3 TSA kit (Perkin Elmer Life sciences) was used as second fluorescent label. Images were acquired with a motorized inverted IX-81 microscope connected to a CCD-FV2T digital camera (Olympus, Aartselaar, Belgium) and processed with LSM Image browser software (Zeiss, Germany).

The Golgi impregnation method was applied with the Rapid Golgi stain kit (FD Neurotechnologies). Briefly, 4-week-old WT and Nestin-Mfp2 -/-mice were anesthetized and cerebella were quickly dissected, cut in the sagittal plane and handled according to the manufacturer's protocol.
Cerebellar halves were embedded in 4% agarose and 100 µm sections were cut on a vibratome, stained and transferred on Superfrost Plus slides. Z-stack images were taken on a motorized inverted IX-81 microscope connected to a CCD-FV2T digital camera (Olympus). PC soma size was determined as described previously (Tavazoie et al., 2005) . Spine density and length were averaged for 3 terminal dendrites for each PC. Spine number was counted and corrected for dendrite length. Spine length was averaged for 6 spines per dendrite and calculated as the distance between the base of de dendrite and the outmost distal point of the spine head. Analyses were performed on 15 PCs per genotype, from 3 WT and 3 Nestin-Mfp2 -/-mice, using the FiJi software.