Evaluating Glial Cell Response in Mice

Mice perfused at 3 days following final rBlast or rSham procedures were further analyzed using immunohistochemistry in coronal sections at levels between +0.5 and −0.5 mm relative to bregma. The CC ROI extended from the midline bilaterally to under the peak of the cingulum.^{21 (link)} Astrocytes were evaluated by immunostaining for glial fibrillary acidic protein (GFAP; polyclonal rabbit anti-GFAP; 1:1000; DAKO, Carpinteria, CA, USA; Agilent Cat# N1506, RRID:AB_10013482). Microglia/macrophages were identified using polyclonal rabbit antibody against ionized calcium binding adaptor molecule 1(IBA1; 1:1000; Wako, Richmond, VA, USA; Cat# 019-19741, RRID:AB_839504). All tissue sections were counterstained with DAPI nuclear stain (Sigma-Aldrich., St. Louis, MO, USA; D9542). For quantification, images within the CC ROI were acquired with a 10 × objective on an Olympus IX-70 microscope using a SPOT RT3 camera. ImageJ software was used to threshold fluorescence levels to quantify the area of immunolabeling above background.^{28 (link)} Images were also acquired with a 40 × objective to examine the morphology of IBA1 immunolabeled cells to compare the density of resting or activated cells.^{20 (link),35 (link),37 (link)} Analysis included six to eight sections per mouse.