Mab3402

Immunofluorescence of retinal sections was performed as previously reported [33 (link), 37 (link)]. The eyecups were cut into 10 μm thick sections. The cryosections were, respectively, incubated with monoclonal mouse anti-mouse A_2AR antibody (05-717; Millipore, USA), polyclonal rabbit anti-mouse Iba-1 antibody (019-19741; Wako, Japan), monoclonal rat anti-mouse CD11b antibody (ab8878; Abcam, USA), monoclonal rat anti-mouse MHC Class II antibody (ab25333; Abcam, USA), monoclonal mouse anti-mouse GFAP antibody (mab3402; Sigma-Aldrich, USA), polyclonal rabbit anti-mouse GFAP antibody (ab7260; Abcam, USA), and polyclonal rabbit anti-mouse IL-1β antibody (ab9722; Abcam, USA) by overnight incubation at 4°C. The cryosections were then, respectively, incubated in Alexa 555-conjugated anti-mouse IgG (4409), Alexa 488-conjugated anti-mouse IgG (4408), Alexa 555-conjugated anti-rabbit IgG (4413), Alexa 488-conjugated anti-rabbit IgG (4412), or Alexa 488-conjugated anti-rat IgG (4416) (all from cell signaling technology, Inc.). The sections were finally counterstained with DAPI (sc-3598; Santa Cruz Biotechnology, Inc.). Images were captured with a confocal microscope (SP5; Leica Microsystems, Inc., USA) with a fixed detection gain for each comparative section.

The mice were perfused with 4% paraformaldehyde in phosphate-buffered saline, and the brain was dissected and placed in sucrose solution. After cryoprotection using a 15 and 30% sucrose gradient, coronal hippocampal slices were prepared at 25-μm thickness using a freezing microtome (CM-1950, LEICA). To confirm the efficiency of the specific PP2A knockout and the effect on the development of neurons and neurogliocytes in the hippocampal CA1 area, the slices were incubated in primary antibody overnight at 4 °C. After incubation with the secondary antibody for 2 h and DAPI (10,236,276,001, Roche, 1 μg/ml) for 15 min at room temperature, the samples were examined by using confocal laser microscopy (FV-1000, OLYMPUS). The antibodies and dilutions were as follows: PP2A C subunit antibody (#2038, Cell Signaling Technology, 1:250), anti-NeuN rabbit polyclonal antibody (ABN78, Millipore, 1:500), goat anti-rabbit IgG (H + L) Cy3 (BS10007, Bioworld Technology, 1:400) and anti-glial fibrillary acidic protein (GFAP) antibody, and clone GA5 (MAB3402, Millipore, 1:500).

Brains were sectioned with a microtome and stained, with every sixth 30 µm section through the injury site labeled with antibodies against CD68 (activated microglia) or GFAP (astrocytes). Sections were imaged by mosaic stitching under a 10× objective. Inflammation was assessed by area and density of CD68 (1:500, Cat #MCA1957, Bio‐Rad, RRID_322219) staining while injury size was determined by a GFAP (Cat #MAB3402, Millipore, RRID_94844) negative area surrounded by GFAP positive astrocytes as described previously (Jia, Malone, et al., 2020 (link)). Both CD68 and GFAP were normalized to staining in the contralateral, non‐injured hemispheres using ImageJ software.

The perihematomal tissues were extracted and then homogenized in lysis buffer containing a protease inhibitor cocktail. The supernatant was collected after centrifugation at 12,000 rpm for 10 min. The protein concentration was determined using the rapid gold BCA protein assay kit (Thermoscientific, Rockford, United States). Equal amount of protein were separated through 10% SDS PAGE then transferred onto a PVDF membrane (Millipore, Billerica, MA, United States). The blots were incubated overnight at 4°C with anti-AQP4 (ab46182, abcam) and anti-GFAP (MAB3402, Millipore) antibodies. Protein bands were detected using an enhanced chemiluminescence detection system (Cell Signaling Technology, Beverly, MA, United States) and the intensities of immunoreactive bands were analyzed using an image analysis program (Image J, NIH, United States).