Supplementary Figure 2: Neonatal hypoxia results in delayed maturation of Purkinje cells and loss of MBP without apoptosis or axonal injury.

(a) Representative confocal images of sagittal sections from normoxic and hypoxic mice (P7-P30) showing calbindin immunofluorescence (green). ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granule cell layer. Scale bars 50 μm. Quantification of Purkinje cell numbers and Purkinje cell layer thickness are presented in Fig. 1d and e. (b) Representative confocal images showing NF200 (green) and MBP (red) immunofluorescence (P7-P30). Images below represent NF200 and MBP overlay merged with DAPI. Abbreviations as in (a), plus WM, white matter. Scale bars 50 μm. Quantification of NF200 and MBP immunofluoresecence is presented in Fig. 1g and h. (c) Immunolabeling of cerebellar sagittal sections from normoxic and hypoxic mice (P11) with calbindin (green) and caspase-3 (red). Images indicate no increase in the expression of caspase-3 in the Purkinje cell layer. Scale bar 50 μm. (d) Representative confocal images of cerebellar white matter sections from normoxic and hypoxic GAD65-GFP mice (P11) labeled with caspase-3. No increase in apoptosis was detected following hypoxia. Apoptotic caspase-3⁺ cells (white arrowheads) are present in the external granule cell layer and in the white matter in both normoxia and hypoxia tissue. Scale bar 25 μm. (e) Confocal images of cerebellar sections from normoxic and hypoxic mice (P11) labeled with the axonal injury marker β-amyolid precursor protein (β-APP) and calbindin. Scale bar 50 μm. A small number of β-APP⁺ cells were seen in the internal granule cell layer of both normoxic and hypoxic mice (white arrowheads). In comparing sections from 5 normoxic and 7 hypoxic mice, no increase in β-APP expression was detected.