C2724

Standard Western blot analysis [4 (link)] of cerebellum lysates (n = 6/time interval, n = 18 control, and n = 18 PNV treated per age) was performed using rabbit polyclonal antibody against CaB (1 : 2000, C2724, Sigma-Aldrich, St. Louis, MO, USA), Flt-1 (1 : 500, sc-316), Flk-1 (1 : 250, sc-315), both from Santa Cruz Biotechnology (Santa Cruz, CA, USA), GAD (1 : 1000, AB1511, Millipore, Billerica, MA, USA) and laminin (1 : 500, L9393, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal antibody against VEGF (1 : 500, sc-7269) and β-Catenin (1 : 600, sc-7963), both from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and β-actin (1 : 1000, A2228, Sigma-Aldrich, St. Louis, MO, USA) and goat monoclonal antibody against occludin (1 : 500, sc-8144, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were visualized using chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA). For quantification, the density of pixels of each band was determined by the NIH Image J 1.45s software (available at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, NIH, Bethesda, MD). For each protein investigated the results were confirmed in three sets of experiments and data were normalized using the respective loading controls. Values were normalized to the corresponding value for β-actin internal control and expressed as a ratio.

At designated time intervals and after anesthesia, the animals (n = 4/time interval, n = 12 control, and n = 12 PNV-treated per age) were perfused transcardially with saline solution followed by 4% paraformaldehyde in 0.1 M PBS (phosphate buffered saline), pH 7.4. The cerebella were dissected out and were embedded in paraffin. Antibodies utilized were anti-VEGF (1 : 50, sc-7269), anti-Flt-1 (1 : 500, sc-316), and anti-Flk-1 (1 : 50, sc-315), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CaB (1 : 1000, C2724, Sigma-Aldrich, St. Louis, MO, USA), and GAD (1 : 500, AB1511, Millipore, Billerica, MA, USA). Immunohistochemistry was performed in sequential coronal 5 μm thick paraffin sections of the cerebellum as previously described [17 (link)]. To avoid procedure differences between control and envenomed groups, the immunohistochemistry for each protein (VEGF/Flt-1/Flk-1 and CaB/GAD) was performed concomitantly. Three images per region (molecular, granular, and Purkinje) of n = 4 rats, totaling 12 images per time/age/treatment, were captured using a BX51 microscope (Olympus Optical C. Ltd., Tokyo, Japan). Objective (20x), lens aperture, and light intensity were set for all images captured.

Immunofluorescent labeling and microscopic imaging has been described previously.16 Primary antibodies used were ßIII‐tubulin (ab78078, 1:250; Abcam, Cambridge, MA), calbindin‐D28K (C2724, 1:500; Sigma‐Aldrich, St Louis, MO), CD11b/c (ab1211, 1:100, Abcam), EGFP (ab6556, 1:500, Abcam), glial fibrillary acidic protein (GFAP; ab33922, 1:200, Abcam), IBA‐1 (ab5076, 1:200, Abcam), NeuN (ab177487, 1:500 and ab104224, 1:500, Abcam), S100 (MAB079, 1:200; Millipore, Billerica, MA), S100 (Z0311, 1:200; Dako, Carpinteria, CA). Secondary antibodies were Alexa Fluor 488/555, goat/donkey anti‐mouse (1:500), Alexa Fluor 488/555, goat/donkey anti‐rabbit (1:500), and Alexa Fluor 488/555, donkey anti‐goat (1:500; Invitrogen, Carlsbad, CA). Sections were mounted in VECTASHIELD medium containing the nuclear dye 4′,6′‐diamidino‐2‐phenylindole (Vector Laboratories, Burlingame, CA).