Chicken anti homer1

For the astrocyte–Homer1 association, free-floating brain sections were immunostained as described above. The following primary antibodies were used: mouse anti-GFAP (1:1,000 dilution, clone ASTRO6, Thermo Fisher), mouse anti-S100B (1:750 dilution, Abcam) and chicken anti-Homer1 (1:1,000 dilution, Synaptic System), followed by incubation with Alexa-conjugated secondary antibodies. Digital images were acquired using a ×100 oil-immersion objective on a Leica SP8 laser scanning confocal microscope or a ×60 oil-immersion objective on an Andor DragonFly spinning disk confocal microscope. Confocal stacks were analyzed using Imaris 9.6.1. For astrocyte–Homer1 associations, the GFAP or S100B channel was subjected to Gaussian filtering and background subtraction. GFAP⁺ or S100B⁺ astrocytes were 3D-reconstructed using the surface-rendering function. Homer1 puncta were reconstructed using the spots function and their total number was calculated. Next, the number of Homer1 puncta located up to 0.3 µm from the astrocyte surface was identified and considered as Homer1 puncta associated with astrocytes. Percentage of astrocyte-associated Homer1 puncta was calculated by dividing it with the total number of Homer1 puncta in each image.

Neuron cultures (35–42 d after differentiation) on glass coverslips were fixed with 4% paraformaldehyde (PFA) and washed with PBS. After blocking and permeabilization with 5% normal goat serum (Vector), 1% BSA (Sigma-Aldrich) and 0.2% Triton- X-100 for 1 h at room temperature, cells were incubated with primary antibodies for mouse-anti-Syn1 (Synaptic Systems), chicken-anti-Homer-1 (Synaptic Systems), and rabbit-anti-MAP-2 (Abcam; Table 1) overnight at 4°C. After washing, secondary labeling was performed using appropriate Alexa Fluor-conjugated goat secondary antibodies (Invitrogen) for 2 h at room temperature. Coverslips were mounted using Vectamount AQ (H-5501; Vector Laboratories).

Coronal brain sections from 2-week-old male and female mice were incubated with rabbit anti-Iba1 (Wako, 019-19741, 1:500) and chicken anti-Homer1 (Synaptic Systems, 160006, 1:500) overnight, followed by Alexa Fluor 594 (703-585-155, Jackson ImmunoResearch, 1:500) and 649 (711-605-152, Jackson ImmunoResearch, 1:500) conjugated secondary antibodies. Images were acquired on a Nikon C2+ confocal laser scanning microscope using a 40× oil objective, laser power and gain were kept consistent throughout the experiment. Z-stack images were acquired with a 0.8-μm interval for 21 steps. Images were analyzed using Amira software (Thermo Fisher Scientific) to create a 3D surface rendering of microglia with a threshold to ensure that microglial processes were accurately reconstructed. This rendering was used to mask the Homer1 channel, and the overlapped volume was considered as Homer1 engulfed by microglia.