Anti cc1

Mice brains were cut into several brain slices (25 µm) by microtome. At room temperature, the brain slides were sealed with 1% immune tissue blocking fluid for 2 h and incubated overnight with rabbit anti-MBP (1:100, Abcam, Cambridge, UK), anti-Iba-1 (1:100, Wako, Japan), anti-CC1 (1:100, Millipore, Burlington, MA, USA), goat anti-Olig2 (1:100, Millipore, Burlington, MA, USA), anti-PDGFRα (1:100, Millipore, Burlington, MA, USA) respectively at 4 °C. Subsequently, the brain slides were incubated with the corresponding secondary antibody at room temperature for 1 h. Then, the corpus callosum site of slices was photographed with a multiphoton laser scanning confocal microscopy system (Olympus Fluoview FV1000) or common fluorescence microscope (Leica DMI6000).

To prepare sections of IUGR and sham brains, rats were killed at postnatal day (PD) 14 by perfusion in 4% paraformaldehyde. Frozen sections were prepared and cut on a Leica cryostat at 12-μm thicknesses, all according to established protocols [21 (link), 22 (link)]. To label mature oligodendrocytes, we used anti-myelin proteolipid protein (PLP) (1:2, rat hybridoma [23 (link)]) and anti-CC1, which labels oligodendrocyte cell bodies (CC1, 1:20, Millipore, Billerica, MA). For microglia/macrophages, we used anti-IBA-1 (Wako Pharmaceuticals, 1:1000) and anti-CD68 (Abcam, 1:100). For IL-4 receptor, we used an anti-IL-4R (Abcam, 1:50). Secondary antibodies of appropriate species and isotype used for external and internal antigens were purchased from Jackson Immunoresearch, West Grove, PA. Coverslips were mounted over the sections in 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA).
To count cells from frozen sections, IUGR and sham animals from at least three litters were used. Digital images were taken at × 20 magnification from sections at the level of the anterior part of the corpus callosum, counting 20,150 μm² regions of interest per section, at least two sections per animal. Statistical significance was calculated using Student’s t test.

Animals were perfused with 4% paraformaldehyde, cryopreserved in 30% sucrose for 48 h, the lumbar region of the spinal cords were embedded in Tissue-Tek OCT Compound (VWR), and 30 µm frozen sections of L2–L4 spinal cords were prepared. For antibody staining, sections were incubated with primary anti-GFAP (Cell Signaling Technology; 1:300), anti-IBA (Wako Chemicals; 1:1000), anti-CC1 (Millipore; 1:300), anti-caspase-8 (Santa Cruz Biotechnology; 1:1000), or anti-NLRP3 (AdipoGen; 1:300) antibodies overnight at 4 °C. Subsequently, sections were incubated with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (1:500, Invitrogen) for 1 h at room temperature. Nuclei were counterstained with Hoechst. Slides were mounted and sections were examined using Zeiss LSM 700 confocal microscope. Maximum projection images from confocal z-stacks were acquired. Care was taken to minimize pixel saturation while imaging each z-stack. Captured images were processed using Zen 2012 Blue acquisition software (Zeiss Inc.). Quantitative analysis of the cells was determined using Image J software. No fluorescence crossover was found between the channel and images were collected separately using appropriate laser excitation. Three–four animals per each group were examined and representative images are shown.