Following seven days upon the treatment, mice were weighed and deeply anesthetized with a ketamine/xylazine cocktail accordingly. Animals were transcardially perfused once with ice-cold PBS 1x (Gibco) and then with 4% paraformaldehyde (PFA). Brains were collected in 4% PFA for an additional 24 h post-fixation and incubated in 20% sucrose for another 24 h.
For immunofluorescence assay, brains were embedded in Tissue-Tek® optimal cutting temperature compound (Sakura), cut into 10 μm sections of thickness using a Shandon Cryotome E cryostat (Thermo Scientific), and mounted on Starfrost® adhesive slides (Knittel Glass). Sections were rehydrated with blocking buffer (10% BSA, 0.3% Triton in TBS), rinsed with TBS (Gibco), and incubated overnight at 4°C with the corresponding dilutions of the antibodies CD45 (BioLegend, clone 30-F11) and CD31 (Santa Cruz, clone M-20) in blocking buffer. After several rinses, sections were incubated with Alexa Fluor 488 (Molecular Probes) or Alexa Fluor 546 (Molecular Probes) antibodies and counterstained with DAPI. Slides were analyzed under a FV1000 laser scanning confocal fluorescence microscope (Olympus). Quantification of microglia numbers and CD45 integrated density area was performed using ImageJ software (NIH, USA).
For immunofluorescence assay, brains were embedded in Tissue-Tek® optimal cutting temperature compound (Sakura), cut into 10 μm sections of thickness using a Shandon Cryotome E cryostat (Thermo Scientific), and mounted on Starfrost® adhesive slides (Knittel Glass). Sections were rehydrated with blocking buffer (10% BSA, 0.3% Triton in TBS), rinsed with TBS (Gibco), and incubated overnight at 4°C with the corresponding dilutions of the antibodies CD45 (BioLegend, clone 30-F11) and CD31 (Santa Cruz, clone M-20) in blocking buffer. After several rinses, sections were incubated with Alexa Fluor 488 (Molecular Probes) or Alexa Fluor 546 (Molecular Probes) antibodies and counterstained with DAPI. Slides were analyzed under a FV1000 laser scanning confocal fluorescence microscope (Olympus). Quantification of microglia numbers and CD45 integrated density area was performed using ImageJ software (NIH, USA).