Alexa fluor 647 conjugated goat anti mouse secondary antibody

Coronal brain slices were cut at a 16 μm thickness on a cryostat microtome (Microm HM 550; Thermo). Sections at the level from −2.3 mm to −4.16 mm from the bregma were chosen by preliminary experiments. Sections were blocked with 5% BSA at 37°C for 1.5 h and then incubated overnight at 4°C with an anti-CD11b antibody (mouse monoclonal, 1 : 200; AbD, USA) along with either an anti-TLR4 antibody (rabbit polyclonal, 1 : 100; Santa Cruz, sc-30002) or an anti-TLR2 antibody (goat polyclonal, 1 : 50; Santa Cruz, sc-16237). Following washing with PBS, the sections were incubated with an Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (1 : 200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1 : 100; Jackson ImmunoLabs), and an Alexa Fluor 488-conjugated donkey anti-goat secondary antibody (1 : 100; Jackson ImmunoLabs) at 37°C for 1 h. Finally, the slides were examined using a Nikon A1R confocal laser-scanning microscope, and positive cells in the hippocampal CA1, CA3, and DG region were captured using NIS elements AR software.

For immunocytochemistry, cultured hippocampal neurons were fixed in 4% paraformaldehyde (PFA) and 4% sucrose directly following Ca²⁺ imaging for 20 min at room temperature (RT). Following fixation, coverslips were washed with PBS + glycine and permeabilized in PBS+ 0.3% Triton X-100 for 20 min at RT. Next, cells were incubated in blocking buffer containing 10% donkey serum (DS; Sigma-Aldrich) and 0.2% Triton X-100 for 1 h at room temperature. For labeling of Shank, cells were incubated in 1:200 anti-Shank primary (NeuroMab; RRID:AB_10672418), 5% DS and 0.1% Triton X-100 for 3 h at RT. Followed by incubation with 1:200 AlexaFluor 647-conjugated goat anti-mouse secondary antibody (Jackson Laboratories) for 1 h at RT. Finally, cells were postfixed in 4% PFA and 4% sucrose for 10 min.