Alexa 488 fluorophore

Whole-mount immunostaining of Ubap2 in the zebrafish larvae was performed as previously described^{58 (link)}. Briefly, 3 dpf larvae were fixed in 4% paraformaldehyde diluted in 1 × PBS with 0.25% Triton X (PBST) at 4 °C overnight. After PBST washing, antigens retrieval was performed using TrisHCl (pH 9.0, 150 mM) at 70 °C for 20 min. The larvae were washed with PBST and then treated with 0.05% trypsin-EDTA for 45 min on ice, and incubated with a blocking buffer (2% normal goat serum, 1% bovine serum albumin, and 1% dimethyl sulfoxide) for 1 h. The larvae were incubated overnight at 4 °C with the primary antibody (Ubap2, 1:10, Abcepta, AP12773a) or without the primary antibody as a negative control and then incubated with a secondary antibody conjugated with Alexa fluorophore 488 (1:500, Invitrogen). Larvae were mounted in 1% low melting agarose and imaged with a confocal microscope (FV1000 confocal microscope, Olympus)^{58 (link)}.

After treatment with LPNP-TLR4
siRNA or LPNP-CD11b siRNA at different concentrations in the Lab-Tek
chamber (155411PK734-2062 vwr), the microglial cells were washed with
PBS and fixed with 1% paraformaldehyde solution for 10 min. After
rinsing the cells with TBS, the TLR4 (Abcam, ab13556, 1:200) or CD11b
(Abcam, ab133357, 1:200) primary antibody was incubated (in SUMI buffer
(2.5 mg/mL gelatin, 0.5% Triton, dissolved in TBS)) with the cells
overnight at 4 °C. After rinsing with TBS, the secondary antibody
(biotinylated anti-rabbit, Vector Lab, BA1100, 1:400) was incubated
with the cells for 1 h at room temperature. The cells were washed
with TBS and incubated with streptavidin Alexa Fluorophore (for TLR4:
streptavidin Alexa Fluorophore 594, Jackson ImmunoResearch, 016-580-084,
1:300; for CD11b: streptavidin Alexa Fluorophore 488, Invitrogen,
s32354, 1:300) for 1 h at room temperature. After washing with TBS,
the cells were incubated with DAPI (Sigma-Aldrich; 1:1000) for 10
min. Following washes in TBS, the fluorescence signals in cells were
detected using a Leica TCS SP5 inverted confocal microscope.

Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were purchased from Sigma-Aldrich (St Louis, MO, USA), NMDA and MK-801 from Tocris Bioscience (Bristol, UK) and Human Ab-oligomers from Anaspec (Fremont, CA, USA) (Cat. No. AS-20276). The human versions of NMDAR subunits NR1, NR2A and NR2B were obtained from Addgene (Watertown, MA, USA) and subcloned to pcDNA3.1. The antibodies used were the following: polyclonal rabbit anti-Nissl (Life technologies, Carlsbad, CA, USA, N21480), Monoclonal mouse anti-Ki-67 (14-5698-82, Invitrogen, Waltham, MA, USA) and polyclonal rabbit anti-Caspase3 (9H19L2, Thermo Fischer Scientific, Waltham, MA, USA), polyclonal rabbit anti-Nectin 3 (ab63931, Abcam, Cambridge, UK) and polyclonal rabbit anti-F-actin antibody fused to an Alexa 488 fluorophore (A12379, ThermoFisher, Waltham, MA, USA). pTau protein was kindly provided by Prof. J. Avila (CBM, UAM-CSIC, Madrid, Spain).

Five antisense DNA probes were designed against the full-length zebrafish sox2 and ntla mRNA sequence as described by Choi et al. (2014) (link). Embryos incubated in either embryo medium were fixed 4% PFA at 4°C and then stained according to Choi et al. (2014) (link). HCR was combined with immunohistochemistry for phospho-histone H3 for identification of mitotic cells at the point of fixation. Embryos underwent HCR followed by blocking in 2% Roche blocking reagent, 5% donkey serum in maleic acid buffer. A mouse anti-pH3 antibody (abcam, ab14955) was incubated overnight at 4° followed by a secondary anti-mouse conjugated to an Alexa 488 fluorophore (ThermoFisher Scientific, A32723). Imaging was performed on a Zeiss LSM700 confocal with identical imaging parameters: z-step 0.5683 μm; 1024×1024 resolution; 63× oil objective NA 1.4; 35% laser power at 488 nm; Gain 661; Digital offset −2; Pixel dwell time 3.12 μs.

Synchronized schizont cultures were magnet-purified and incubated in E64 (10 µM) for 3–5 hours until fully segmented. Parasites were passed through a 1.2 μm syringe filter to obtain merozoite suspension. Merozoites were mixed with erythrocytes in culture media supplemented with dimethyl sulfoxide or R1 peptide (100 µg/ml) and incubated at 37°C with shaking for 1 min 30 s until fixing. Parasite cultures were spun down at 2000×g and fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate-buffered saline (PBS) for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 25 min, and incubated in blocking solution (2% bovine serum albumin in PBS) for 1 hour at room temperature. Cell suspensions were then mounted onto 1% polyethyleneimine-treated coverslips, the excess liquid was removed and samples were mounted on slides with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI). The primary antibody used to detect the C-terminus of RON3 was rat anti-HA (3F10, Roche, 1:500) and the secondary antibody conjugated to Alexa-488 fluorophore from Thermofisher (1:1000). Wheat Germ Agglutinin 647 conjugate (Thermofisher) was used to stain the erythrocytes at 1:1000 dilution.