Evos fluorescence microscope

Cells were fixed immediately with fixing solution (2% paraformaldehyde, 100 mM KCl, 200 mM sucrose, 1 mM EGTA, 1 mM MgCl₂, 10 mM PIPES, pH 6.8) for 10 min and then treated with permeabilization buffer (0.2% Triton X-100, 100 mM KCl, 200 mM sucrose, 1 mM EGTA, 1 mM MgCl₂, 10 mM PIPES, pH 6.8) for 10 min after washing with PBS. The cells were then washed three times with PBS and incubated for 15 min with blocking solution containing 3% BSA in PBS. The cells were then washed a further three times with PBS and incubated overnight with the primary antibodies in blocking solution. Antibodies to OCT4 and H3K27me3 were purchased from Cell Signaling Technology. Next day, the cells were washed three times with PBS and incubated with the secondary antibody, a goat anti-mouse antibody conjugated with Alexa Fluor® 488 fluorescent dye (Molecular Probes, Eugene, OR), in blocking solution. After further washing with PBS, the cells were stained with Hoechst dye to stain the nuclei. Each image was examined using an EVOS fluorescence microscope (Invitrogen).

As previously described (Han et al., 2020 (link)), mice were anesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS), and tissues were fixed in 4% PFA at 4°C for 12 h. After dehydration by 30% sucrose, brain tissue was cut into 30-μm-thick sections using a freezing microtome (Leica CM30505, Germany). The posterior portion of the BLA-containing sections was permeabilized with 0.3% Triton X-100 and 5% donkey serum in PBS for 2 h and then incubated with rabbit polyclonal anti-c-Fos antibody (1:1,000, Abcam, Cambridge, United Kingdom, ab190289) or rabbit polyclonal anti-PV antibody (1:1,000, Abcam, ab11427) at 4°C overnight. After washing three times with PBS, sections were incubated with Cy3-labeled anti-rabbit secondary antibody (1:1,000, Absin, Shanghai, China, abs20024) for 1.5 h at room temperature and then given three 10-min washes in PBS. Afterward, sections were incubated with DAPI solution (1:5 for 15 min, Beyotime, Shanghai, China, C1005) for nuclear labeling and coverslipped with anti-fade mounting medium. Images were captured using an EVOS fluorescence microscope (Invitrogen, United States). For c-Fos immunostaining, mice subjected to several behavioral tests were perfused 1.5 h after the tests.

HEK 293T cells were grown in complete media (high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium supplemented with 10% foetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin) and maintained in a humidified incubator at 37 °C and 5% CO₂. Prior to transfection, cells were seeded at a confluency of 40% in 48-well plates. Cells were transfected with 200 ng of saRNAs using commercially available liposome-based Lipofectamine™ MessengerMAX™ transfection reagent (Invitrogen, Waltham, MA, USA) at an RNA (µg): Lipofectamine™ (µL) ratio of 1:1.5, or LNP formulations, as described in Section 3.3, Section 3.4 and Section 3.5. The expression of fluorescent reporter proteins was examined at various timepoints using an EVOS Fluorescence Microscope (Invitrogen, Waltham, MA, USA). The expression of Luc2 was examined using a Luciferase^® Reporter Assay System kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, at 24 or 48 h post-transfection, cells were lysed by incubation in passive lysis buffer with shaking for 15 min at room temperature. LARII substrate was added to cell lysates and relative light units were detected using GloMax^® Explorer (Promega, Madison, WI, USA).

NHDF cells infected with tachyzoites of T. gondii at a ratio of 5:1 of parasites to host cells were treated with 1 µM clindamycin, MMV 1634391, and retapamulin for 24 h. After treatment, cells were fixed with 3.7% freshly prepared formaldehyde, and prepared as previously described [32 (link)]. Anti-CPN60 (kindly provided by Dr. Boris Striepen, University of Georgia, USA) was used to label the apicoplast luminal protein CPN60 at a dilution of 1:2000. DAPI (5 µg/mL; Sigma-Aldrich) was used to label the DNA. After labeling, the coverslips were mounted onto slides using Prolong gold (Life Technologies, Eugene, OR, USA), and samples were examined on an Invitrogen EVOS fluorescence microscope.