Lsm880 axioobserver z1

Immunofluorescent (IF) staining for brain tissue was performed as previously described [44 (link)]. Embryonic brains were collected at indicated time points after in utero electroporation (IUE). Brains were immersed in 4% paraformaldehyde for 1 day and incubated in 20% sucrose for 2 days at 4 °C. Sectioned brain at a thickness of 14 μm on a Microm cryostat microtome (Thermo Fisher Scientific, MA, USA) was permeabilized with 0.25% triton X-100 for 10 min, then incubated in 10% normal horse serum for 1 h for blocking. The blocked sections were incubated with mouse anti-GFP (diluted 1:400) overnight. Sections were thoroughly rinsed in PBS and incubated with Alex-488-conjugated anti-mouse IgG (1:200). All photomicrographs were taken using Zeiss LSM880 AxioObserver Z1 with AiryScan FAST (Oberkochen, Germany) or Zeiss Axoimager M2 (Oberkochen, Germany). Leading process of GFP-positive neuron on cortical plate was used for measurement of axonal length as described [6 (link)].
Axonal length from neuronal cell culture was measured at 3 days after plating and the GFP positive longest neurite of primary neurons was defined as the major axon as previously described [6 (link)]. The measurement of axonal length was carried out using Zeiss ZEN imaging software (Oberkochen, Germany).

Following 1 or 24 h of infection, the macrophages were washed with PBS, fixed in 2% paraformaldehyde for 30 min, washed once with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. We stained nucleic acids with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Sigma), and finally, the cells were washed three times with PBS. The slides were observed using an inverted Zeiss LSM 880 Axio Observer Z1 confocal laser-scanning microscope with an Airyscan detector. Cells were visualized using a Zeiss Plan-Apochromat oil lens objective of 63× and a numerical aperture of 1.4. Z-stacked images were acquired with a digital zoom of 8× (1.8× for broad fields), using the Zen lite software (Carl Zeiss microscopy). Images were processed using the Image J software package. A single representative section of the compiled Z-projections produced by Image J software is presented in all the figures.