Glial fibrillary acidic protein (gfap)

Immunocytochemistry was performed as described previously [27 (link)]. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. After blocking with 10% donkey serum, fixed cells were incubated with primary antibodies (Iba1, 1:1,000, WAKO Chemicals; GFAP, 1:1,000, Abcam) for 2 h followed by fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 and 555, 1:200, Molecular Probes, respectively). Nuclei were counterstained with DAPI. Fluorescence images were acquired using a confocal-laser microscope (LSM 700; Carl Zeiss MicroImaging) with a multi-track configuration.
For immunohistochemistry, WT and P2X7^−/− aged matched mice were perfused. Brains were dissected out, cryo-protected, and cut. Brain sections were stained with primary antibodies (P2X7, 1:500, Sigma; Iba1, 1:500, Abcam; GFAP, 1:500, Abcam) for 48 h at 4 °C followed by fluorochrome-conjugated secondary antibodies (Alexa Fluor 488, 647, and Cy3, 1:500, Jackson Laboratory, respectively). Nuclei were counterstained with Hoechst. Images were acquired using a confocal-laser microscope (LSM 700; Carl Zeiss MicroImaging) and displayed with maximum projection of z-stacks.

For immunohistochemical analysis of STAT3 and GFAP the paraffin embedded brains were sectioned with a manual rotary Microm microtome (Thermo Scientific, Kalamazoo, MI) into 6 µm sections and floated onto Superfrost + slides (Fisher Scientific). The slides were warmed at 60°C for 20 min. To dissolve away the paraffin, slides were put through xylene and then 100% followed by 95% ethanol washes. Following an overnight incubation in primary antibody STAT3 (rabbit;1∶300; Cell Signaling Technology, Inc., Danvers, MA); GFAP (chicken;1∶300; Abcam, Cambridge, MA) sections were rinsed in PBS and incubated with secondary antibody (for STAT3, 1∶250; for GFAP 1∶250) for 4 hours at room temperature. The sections were rinsed and coverslipped with prolong gold antifade with DAPI mountant (Life Technologies, Grand Island, NY). The sections were visualized using an Olympus AX70 microscope with a PlanApo 40x 0,85 NA objective lens and images captured using Cell Sens Dimension software with the Olympus DP73 digital camera attached to the microscope. Post-processing of images was done according to accepted practices and image integrity guidelines (e.g., [34] , [35] ). Specifically, the tone was normalized in all images and STAT3 images were sharpened.

After animals were sacrificed, mice were perfused with saline and sequenced 4% paraformaldehyde fixation solution through the left ventricle and then quickly frozen in precooled isopentane. The brain was coronal sectioned to a 20-μm-thick slice. For immunofluorescence staining, sections were rinsed three times by PBS for 5 min, blocked by 5% normal donkey serum for 60 min, incubated with GFAP (1:300 dilution, Millipore Inc., Billerica, MA) and NT-1 (1:200 dilutions, Abcam, CA) antibodies at 4 °C overnight. After rinsing, sections were incubated with IgG for 60 min. For double immunofluorescence staining, sections were pretreated as described above; antibodies for DCC (1:100 dilutions, Santa Cruz, CA) and GFAP, A2B (1:200 dilutions, Santa Cruz) and GFAP, and UNC5H2 (1:100 dilutions, Abcam, Shanghai, China) and GFAP were incubated together; and corresponded secondary antibodies were incubated individually. The pictures were taken under confocal or fluorescent microscopes.