448 Immunocytochemistry The procedures follow those described previously (Morel et al. 1997, 2002; Morel 2007). Prior to be exposed to antibodies, some series were pre-treated for 10 min by exposure to microwave in a 4% aluminium chloride solu- tion (Evers and Uylings 1994) to improve antigen retrieval in formalin stored sections. All series for immunocyto- chemistry were preincubated 10 min in 1.5% H2O2 to remove endogenous peroxidase activity. After several rin- ses in phosphate buffered saline (pH 7.4), sections were incubated 48 h at 4 C or 24 h at room temperature in primary antibodies (dilutions 1:1,000–1:5,000; depending on the antibody and tissue ﬁxation), 2% normal horse or goat serum; and 0.2% triton-X-100. The antibodies used were mouse monoclonal antibodies directed against par- valbumin (PV) or calbindin (CB) (SWant, Bellinzona, Switzerland or Sigma, St Louis, MO, USA), rabbit poly- clonal against calretinin (CR) (SWant), mouse monoclonal against the nonphosphorylated neuroﬁlament protein (SMI- 32; Sternberger Monoclonals Inc., Covance Research Products Inc., Princeton, USA). After several rinses, sections were incubated 30–60 min at room temperature in biotinylated secondary antibodies (1:200, Vector Laboratories, Burlingame, CA, USA) and stained with the avidin–biotin complex (ABC) immunoperoxidase method (Vectastain Elite kits, Vector Laboratories). The reaction was visualized with 3,30-diaminobenzidine tetrahydro- chloride as chromogen, diluted 0.05% in 0.05 M Tris–saline (pH 7.7) and 0.001% H2O2. As controls, the Brain Struct Funct (2008) 212:443–463 primary antibody was omitted while the rest of the proce- dure remained the same. Data analysis Contours of Nissl stained sections were drawn with a Wild stereomicroscope (Leica) equipped with a camera lucida, or traced on scanned images of the sections using Adobe Photoshop (version CS2) and Adobe Illustrator (version CS2). Then, adjacent sections were superimposed using contours and blood vessels. The different ﬁbre tracts (pal- lidothalamic, cerebellothalamic, and medial lemniscus/ spinothalamic) were delineated on myelin stained sections and the contours superimposed on multiarchitectonic and stereotactic maps of the thalamus and basal ganglia (see Morel 2007 for graphical representations, parcellations and nomenclature). We adopted a Latin nomenclature for the cerebello- and pallidothalamic tracts, i.e. fasciculus cere- bellothalamicus (fct) for the ﬁrst; and fasciculus lenticularis (ﬂ), fasciculus thalamicus (ft), and ansa len- ticularis (al) for the second. The delimitation was based on similar criteria for all cases, i.e. corresponding to the thick part of the tract (or ‘‘bulk of the tract’’, as shown in Fig. 3a, c), whether at pallidal or subthalamic level. The entrance into the thalamus was traced for only part of the tract in clear continuity with the prethalamic ﬁbres. Adjacent sec- tions processed for AChE, CaBP or SMI-32 provided additional criteria for ﬁbre tract delimitation, as well as for the subcompartmentalisation of the basal ganglia and Fig. 3 Representation of the cerebellothalamic (fct) and pallidothalamic (al, ﬂ, ft) tracts on sagittal section of the atlas (panel c) as delimited from myelin (area comprised in the rectangle in a). Thalamic nuclei and STh were best identiﬁed on adjacent Nissl section (panel b). Panel d shows PV immunostaining at same sagittal level. The arrows in a and d point to a small gap separating the fct and ft, visible in myelin and PV-ir, respectively. Notice some PV-ir enhanced ﬁbres in the internal capsule near the anterior pole of the STh and presumably corresponding to the fasciculus subthalamicus. Scale bars (a and c): 2 mm 123 a PuM SC bic IC CL MDpl LD CM Pf ml VPMpc fct ft SNc SNr CL VLpd VLpv VM ft ZI STh mcl VApc R VLa fl ot al L 8.1 ac b CM c CM d VPMpc fct ft VLpv fct ft VLpv VLa VLpv VLa VApc ZI STh VM ft STh STh mcl Nissl al PV ZI fl DV 0