Plan apochromat

Immunohistochemistry was performed as previously described^{12 (link), 27 (link), 41 (link)}. Briefly, brains were fixed in 4% paraformaldehyde at E16.5 and coronally sectioned with a cryostat (Leica Microsystems) at a thickness of 60 μm. The slices were incubated with primary antibodies diluted in PBS containing 1% bovine serum albumin (BSA) and 0.01% Triton X-100 at 4 °C overnight followed by Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies diluted in PBS containing 1% BSA and 0.01% Triton X-100 for 1 h at room temperature. The nuclei were visualized by staining with Hoechst 33342 (Invitrogen). Confocal images were recorded using an LSM 780 built around an Axio Observer Z1 with Plan-Apochromat 20 × (numerical aperture (NA) 0.75), C-Apochromat 40 × (NA 1.2), or Plan Apochromat 63 × (NA 1.40) lenses under the control of LSM software (Carl Zeiss). Coronal sections of cerebral cortices containing the labeled cells were classified into CP and IZ, which were outer layer and middle layer, respectively. The number of all labeled cells in each region was calculated. To evaluate the morphology of the migrating neurons, projection images of EGFP-positive neurons were obtained from Z-series confocal images using LSM software. At least three independent fetal brains were electroporated and analyzed for each experiment. All experiments were performed in a blinded manner.

The number of axonal terminals perforating PNNs was quantified in 33 × 33 × 5 µm z-stacks obtained with high-resolution confocal microscopy using the LSM 710 microscope (Zeiss, 100 × alpha Plan-Apochromat objective, NA 1.46, voxel size 60 × 60 × 500 nm). ROIs were positioned in the motor cortex L5 (see Additional file 4: Fig. S1) containing a single PNN-coated neuron. Synaptic terminals expressing VGAT or VGLUT1 that associated with WFA labeling were counted using an automated ImageJ routine (see Additional file 1). In each animal, four ROIs obtained from two adjacent brain sections were analyzed.
The surface of microglia/macrophage-neuron contacts was quantified in 75 × 75 × 10 µm z-stacks obtained with high-resolution confocal microscopy using the LSM 710 microscope (Zeiss, 63 × alpha Plan-Apochromat objective, NA 1.4, voxel size 70 × 70 × 450 nm). ROIs were positioned in the motor cortex L5 containing a single PNN-coated neuron. Surfaces representing IBA1 (microglia/macrophages) and Kv3.1 (fast-spiking interneurons) labeled cells were generated by automated thresholding with IMARIS 9.9 software (Oxford Instruments, Stockholm, Sweden) using the standard surfaces function. The area of contact between cells was quantified as the intersection between IBA1 and Kv3.1 surfaces.

All images (spatial resolution of 1024 × 1024) were acquired under a laser-scanning confocal microscope (LSM510 Meta, Zeiss) with a 20× air (Plan Apochromat, 0.75 NA, Zeiss) or 40× oil-immersion objective lens (Plan Apochromat, 1.3 NA, Zeiss). For imaging whole-mount stained retinas, we acquired confocal z-stacks obtained through the GCL and INL. To compare MEGF10 immunofluorescence signal between WT and KO samples, retinal slices obtained from four pairs of P5 WT and KO retinas were processed for immunostaining at the same time to minimize experimental variations. Images were acquired under the same exposure condition, which yielded the fluorescence intensity of ChAT and MEGF10 in the regions of interest (ROIs) with a given value ≤255 at each single pixel. The ROIs (i.e., soma and dendritic stratification of ON and OFF SACs) were selected manually and analyzed by using ImageJ v1.47. The mean fluorescence intensity of MEGF10 was quantified and normalized to that of ChAT by using ImageJ v1.47. In total, 24 images were analyzed for each genotype, with six images taken from each retinal slice. The total selected ROI areas in WT-ON SAC, KO-ON SAC, WT-OFF SAC and KO-OFF SAC are 67296, 65304, 54316 and 52774 μm², respectively. To measure the diameter of SACs, these images were threshold adjusted to mark each cell, followed by particle analysis using ImageJ v1.47.

The MPM imaging system is a commercial device combined with a Zeiss LSM 880 laser scanning microscope and a mode-locked Ti : sapphire laser, tunable from 690 nm to 1064 nm. In our experiments, the excitation light of 810 nm was chosen for MPM imaging. In channel mode, images can be collected in two separate channels simultaneously. One channel of detecting SHG signals (green color coded) was set from 395 nm to 415 nm, whereas the other channel of detecting TPEF signals (red color coded) was set from 428 nm to 695 nm. In lambda mode, spectral images were obtained by collecting emission signals between 389 and 716 nm. In order to obtain clear large-area images, we chose a 20x (Plan-Apochromat, NA = 0.8, Zeiss, Jena, Germany) objective in this work for channel mode and chose a 63x (Plan-Apochromat, NA = 1.4, Zeiss, Jena, Germany) oil immersion objective for lambda mode.

The expression of GFAP and IBA1 markers was analyzed using an AxioObserver Z1 microscope (objective Plan-Apochromat 10 × /0.45 M25; Zeiss). In the whole-section images obtained by tiling, 600 × 600 µm regions of interest (ROIs) were selected in the motor cortical layer 5 as shown in Fig. 1E, G, and the mean pixel intensity was measured using ImageJ (National Institutes of Health, Bethesda, MD, U.S.A.). In each animal, four images obtained from two adjacent brain sections were analyzed in the ipsilesional and contralesional motor cortex.
The cell density of interneurons expressing PV, Kv3.1, and PNNs was quantified manually in 600 × 600 × 10 µm ROIs obtained in the motor cortical L5 regions using the LSM 710 confocal microscope (Zeiss, 20 × Plan Apochromat objective, NA 0.8, pixel size 0.21 µm). In each animal, four image stacks obtained from two adjacent brain sections were analyzed in the ipsilesional and contralesional motor cortex.