Tuj 1

The western blot technique was performed in cerebral cortex tissue lysates, as previously described [28 (link)]. The primary antibodies used were PLP (proteolipid protein), MBP (myelin basic protein), major histocompatibility complex class II (MHC-II), CD11b (Abcam, Cambridge, UK), synaptotagmin, synapsin IIa (BD Bioscience, Madrid, Spain), Tuj-1 (Neuromics, Minneapolis, USA), and caspase 3 (active fragment of 17 kDa) (Cell Signaling, Massachusetts, USA). Membranes were washed, incubated with the corresponding HRP-conjugated secondary antibodies, and developed with the ECL system (ECL Plus; Thermo Fisher Scientific, Illinois, USA). All the membranes were stripped and incubated with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control (Chemicon, California, USA). Band intensity was quantified with the ImageJ 1.44p analysis software (National Institutes of Health, USA). The densitometry analysis is shown in arbitrary units normalized to the loading control. Additional file 1: Table S1 shows that the basal values of the various proteins evaluated by western blotting in the pup’s cortices display no significant differences when comparing both the WT and TLR4-KO groups.

Cells were fixed with 4% paraformaldehyde, rinsed three times with phosphate buffered saline (PBS) and permeabilized with cold 100% methanol. Cells were stained with a primary mouse antibody to neuron-specific class III beta-tubulin (Tuj-1, Neuromics, Edina, MN, 1:100 dilution) overnight at 4°C. After primary antibody incubation, cells were rinsed three times with PBS and stained with secondary antibodies for one hour at 37°C. The secondary antibody used was a goat anti-mouse linked to Alexa Fluor 488 (1:100 dilution, Life Technologies, Eugene, OR). After secondary antibody incubation, the cells were rinsed three times with PBS. Nuclei were stained with diamidino-2-phenylindole (DAPI, ThermoFisher, 300 nM) for 20 minutes and then rinsed with PBS before imaging.

After organotypic culture, the explants were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 in PBS, and immersed in blocking solution at room temperature for 1 h. The samples were then incubated with different primary antibodies: Tuj 1 (1 : 1000, Neuromics, USA), Neu N (1 : 1000, Cell Signaling Technology, USA), cleaved-caspase 3 (1 : 400, Cell Signaling Technology, USA), PY489-β-catenin (1 : 400, DSHB, USA), and C-myc (1 : 800, Cell Signaling Technology, USA) diluted in blocking solution at 4°C overnight. The next day, the samples were incubated with FITC-conjugated, TRITC-conjugated, or Cy5-conjugated (1 : 1000, Invitrogen, USA) secondary antibody along with DAPI (1 : 3000, Sigma-Aldrich, USA) at room temperature for 1 h. Then, the coverslips were mounted and the samples were observed under a laser scanning confocal microscope (Leica, Germany).