Wild type C57BL/6 mice were subjected to SAH and sham surgery with and
without neutrophil depletion and sacrificed at specific time points after
surgery. Brains were fixed in 4% paraformaldehyde for 24 h at
4°C after perfusion with PBS. They were cryoprotected in 30%
sucrose at 4 °C overnight and embedded in OCT solution and sectioned
into 40-μm cryosections. Free floating sections were incubated for 45
min in TBS containing 0.1% Triton X-100 and 5% normal goat serum
(NGS) and incubated with monoclonal antibody Ly6G (for neutrophils) or Iba-1
(Waka labs, 1:1,000 dilution)] overnight at 4 °C. Sections were
incubated in Alexa Fluor 488–goat anti-rabbit IgG (Invitrogen; 1:500
dilution) and Alexa Fluor 594–goat anti-mouse IgG (Invitrogen; 1:500
dilution) for 1 h at room temperature, rinsed, and air dried before mounting.
Confocal imaging was done using Leica DM 6000 CSF confocal microscope with Leica
Microsystems LAS AF-TCS SP5.
C57BL/6 EGFP-LysM (Lyz2tm1.1Graf) mice were
subjected to SAH and slices were evaluated 3 days after SAH using the same
preservation and staining technique with the exception that the staining was
performed in the dark to prevent loss of fluorescent signal. A secondary
anti-GFP antibody (ThermoFisher Scientific, Waltham, MA) was used to enhance the
signal. Confocal imaging was done using Leica DM 6000 CSF confocal microscope
with Leica Microsystems LAS AF-TCS SP5.
without neutrophil depletion and sacrificed at specific time points after
surgery. Brains were fixed in 4% paraformaldehyde for 24 h at
4°C after perfusion with PBS. They were cryoprotected in 30%
sucrose at 4 °C overnight and embedded in OCT solution and sectioned
into 40-μm cryosections. Free floating sections were incubated for 45
min in TBS containing 0.1% Triton X-100 and 5% normal goat serum
(NGS) and incubated with monoclonal antibody Ly6G (for neutrophils) or Iba-1
(Waka labs, 1:1,000 dilution)] overnight at 4 °C. Sections were
incubated in Alexa Fluor 488–goat anti-rabbit IgG (Invitrogen; 1:500
dilution) and Alexa Fluor 594–goat anti-mouse IgG (Invitrogen; 1:500
dilution) for 1 h at room temperature, rinsed, and air dried before mounting.
Confocal imaging was done using Leica DM 6000 CSF confocal microscope with Leica
Microsystems LAS AF-TCS SP5.
C57BL/6 EGFP-LysM (Lyz2tm1.1Graf) mice were
subjected to SAH and slices were evaluated 3 days after SAH using the same
preservation and staining technique with the exception that the staining was
performed in the dark to prevent loss of fluorescent signal. A secondary
anti-GFP antibody (ThermoFisher Scientific, Waltham, MA) was used to enhance the
signal. Confocal imaging was done using Leica DM 6000 CSF confocal microscope
with Leica Microsystems LAS AF-TCS SP5.