Hoechst 34580

For measurement of mitochondrial membrane potential (MMP), cells cultured at low density on 24 well glass-bottom plate were incubated for 20 min at 37 ℃ with the following probes: 40 nM tetramethyl rhodamine methyl ester (TMRM, Molecular Probes, Invitrogen, Carlsbad, CA, USA), 1 μM Mito Tracker Green (MTG, Molecular Probes) and 2 μM Hoechst 34,580 (Molecular Probes) to monitor MMP. The fluorescence value was detected at wavelength by enzyme labeling instrument^{27 (link)}.

Cryosections on slides were warmed to room temperature for 10 min and fixed in 1:1 methanol/acetone at -20°C for 5 min. After rinsing in phosphate-buffered saline (PBS) containing 1% Triton X-100 (PBST), the sections were blocked with PBS containing 10% fetal bovine serum at room temperature for 90 min. Subsequently, the sections were incubated overnight (4°C) with primary antibodies against these proteins: Pax7 (1:20 dilution; Developmental Studies Hybridoma Bank, Iowa, USA); and desmin (1:200), myosin heavy chain (MHC; 1:50), and collagen type IV (ColIV, 1:400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were then rinsed in PBST, incubated with either fluorescein- or rhodamine-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, Baltimore, MD, USA), counterstained with Hoechst 34580 (Molecular Probes, Eugene, OR, USA), and mounted with fluorescence mounting medium (DakoCytomation, Carpinteria, CA, USA) before being examined using a fluorescence microscope. To detect proliferating cells in tissues, 100 mg/g of 5-ethynyl-2´-deoxyuridine (EdU) was intraperitoneally injected into animals 24 h before harvesting tissues. On the sections, EdU was detected by using the reagents provided in the Click-iT EdU Alexa Fluor 594 Imaging Kit (Invitrogen) according to manufacturer’s instructions. EdU labeling was performed before immunofluorescence staining.

This was performed as previously described [36 (link)]. In brief, de-waxed and washed Methanol-Carnoy fixed tissues were hybridized with the general bacterial probe, EUB338-I (250 ng, 5’-GCT GCC TCC CGT AGG AGT-3’, Eurofins genomics, Ebersberg, Germany), conjugated to Alexa Fluor 555 (Molecular Probes, Thermo Fisher) [11,36]. For mucus visualization slides were incubated with a rabbit anti-MUC2-C3 antibody (1:500) followed by incubation with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000, Molecular Probes, Thermo Fisher). Nuclei were stained with Hoechst 34580 (Molecular Probes, Thermo Fisher) at 1 μg/ml in PBS. Slides were dried and cover-slipped with ProLong Antifade (Molecular Probes, Thermo Fisher). Micrographs were obtained using an Eclipse E1000 (Nikon) fluorescence microscope with the NIS element software (Nikon).

Cells were seeded on coverslips and were fixed with 4% paraformaldehyde for 30min at room temperature. After permeabilization with 0.2% TX-100 (Sigma, X100) for 10min at room temperature, cells were incubated with cleaved caspase 3 antibody (ab13847), cathepsin B antibody (ab33538) or TFEB antibody (sc-48784) for 2h at room temperature. After washing with PBS, cells were incubated with Rhodamine RedTM-X goat anti-rabbit IgG (Molecular Probes, R6394) and with Hoechst 34580 (Molecular Probes, H21486) for 1h. Immunofluorescence images were viewed under Olympus FluoView1000 (FV1000; Olympus) with identical acquisition parameters for the same image session and analyzed with Olympus FLUOVIEW Ver1.7a Viewer. Cleaved caspase 3 and TFEB nuclear staining were quantified with the ImageJ software. Briefly, nucleus of cell of interest was defined using the drawing tool. Mean fluorescence of the nuclear staining was obtained by selecting the “measure” option. After deducting any background staining, the mean fluorescence of at least 50 cells from each treatment was used for statistical analysis. Data is represented as mean +/- SEM (standard error of mean).

We used the adherent indifferenciated NG108-15 cell line which is a transformed hybrid cell line developed by Bernd Hamprecht in 1971 and derived from the union of rat glioma cells with cancerous mouse neuroblastoma cells in the presence of inactivated Sendai virus (Daniels and Hamprecht, 1974 (link)). NG108-15 cells have been extensively used as a model for investigating the process of neuronal differentiation in in vitro studies (Ingraham and Maness, 1990 (link); Beaman-Hall and Vallano, 1993 (link); Tojima et al., 2000 (link); Sukma et al., 2005 (link)). The cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Biochrom, with 4,500 mg/l glucose, L-glutamine, without sodium pyruvate) supplemented with 10% fetal calf serum FCS (Biochrom) and 1% 10,000 U/ml penicillin/streptomycin (Biochrom). For live staining of filamentous actin, 1 μM staining solution was obtained by diluting 1:1,000 1 μl of SirActin stock solution (Spirochrome) with the cell medium. 0.1 mg/ml Hoechst-34580 (Invitrogen/Molecular Probes) was used for nuclear staining. These two stains were used since the actin cortex displays the cell outline and the nucleus is a prominent structure for cell tracking.

Isolated microglial cells on coverslips were incubated with 2 μg/mL FITC-tagged OX42 antibody at 37°C, fixed after 5, 10, 20, and 60 min and nuclei stained with Hoechst 34580 (Molecular Probes®; 1:500, 10 min). Control cells were incubated for 30 min on ice. Non-specific internalization of antibody was examined by incubating microglia with Alexa 488-labeled X63 antibody (60 min, 37°C). The average green fluorescence was measured in the perinuclear region of each cell after background subtraction. Three independent experiments were performed (n ≥ 77 cells per condition). Antibody internalization was quantified (ImageJ) as the increase in intracellular fluorescence by accumulated OX42 antibody in the perinuclear region. One-way analysis of variance (one-way ANOVA) and post hoc one-sample t-tests with Bonferroni correction were used to assess significance.

The sections were incubated with rabbit monoclonal anti-β-catenin (ab32572, 1:250) at 4°C overnight and then incubated with Alexa^® 488-conjugated goat anti-rabbit secondary antibody (Thermo Fisher, Waltham, MA, United States). The nuclear stain Hoechst 34580 (5 μg/ml; Molecular Probes, Thermo Fisher, Waltham, MA, United States) was added prior to final washes after the incubation of secondary antibody. Images were collected via an Olympus confocal laser scanning microscope. DAPI was used for nuclear counterstaining.