Mowiol solution

After discarding the medium, mock- and virus-infected cells were fixed. The TUNEL assay was carried out as described previously [10 (link)]. For PI staining at 30 min before the desired interval, the growth medium was changed to DMEM containing 10 µg/mL of PI (Sigma). Cells were fixed with 3.7% of paraformaldehyde solution in phosphate-buffered saline (PBS) for 15 min at RT, then washed three times in PBS. Nuclei were stained with Hoechst 33342 (Sigma, 1 µg/mL) for 15 min at RT. Coverslips with cells were placed onto a drop of Mowiol solution (Sigma) on slides.

Macrophages isolated from wild type, TLR2^−/− and TLR4^−/− mouse were grown overnight on coverslips and then incubated for 30 min at 37°C with Rv1016c (20 μg/ml) in Hank's buffer. For flow cytometer, after washed with PBS and fixed for 15 min using 4% paraformaldehyde, the cells were stained with mouse anti-His IgG conjugated with Alexa 488 (Santa Cruz Biotechnology), then the cells were collected and 10,000 total events per sample were analyzed by using a BD FACScan calibrator (BD Biosciences, San Jose, CA). For confocal microscopy assay, macrophages were stained with anti-Rv1016c and anti-mouse TLR2 for 2 h at 37°C in PBS containing 2% BSA. After washing, the cells were stained with appropriate Alexa 488− or Alexa 555-conjugated secondary antibodies for 1 h at 37°C, and then they were incubated with 0.1 μg/ml DAPI for 5 min at room temperature. Finally, cells were mounted on slides using Mowiol solution (Sigma-Aldrich) and observed using a 63x oil objective on an LSM 710 microscope (Carl Zeiss, Munich, Germany).

For immunocytochemistry experiments cells were fixed 24 or 48 h after the SW and treatment in 4% formaldehyde solution (PFA), 20 min at RT. If the antibody for the target protein was against extracellular protein domain, the cultures were firstly blocked (5% BSA in PBS) for 1 h, at RT, then incubated with selected antibody of interest, followed by appropriate secondary fluorophore-tagged antibody. List of all used primary and secondary antibodies and their concentrations are given in Table 1. Subsequently, the cells were permeabilized (0.05% Triton in PBS) for 15 min, at RT, for further protein targets fluorescence labelling. All primary antibodies were incubated overnight, at +4°C, while the fluorescent secondary antibodies were incubated for 2 h, at RT. Cell nuclei were stained with Hoechst (2 ug/ml final dilution, for 15 min, at RT, Cat. no. 62249 Thermo Fisher Scientific, United States). Glass coverslips were mounted on microscope slides with MOWIOL solution (Sigma-Aldrich, St. Louis, Missouri, United States). The omission of the primary antibodies resulted in the absence of any specific immunoreactivity.

Cells were cultured on glass coverslips and fixated with 10% formalin solution (Sigma-Aldrich) for 10 minutes at room temperature. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS, and stained with primary antibodies for 1 hour followed by secondary antibodies for 30 minutes. For blocking, the antibodies were diluted in room-tempered DMEM with 10% FBS upon staining of coverslips. The coverslips were washed three times in PBS after fixation, permeabilization and antibody incubations. Coverslips were mounted with mowiol solution (Sigma-Aldrich). Antibodies are listed in Supplementary Table 2.

After 48 h of infection with M. agalactiae, HeLa cells were washed with PBS to remove any serum content, and the cells were permeabilized by incubation with 70% alcohol for 1 min. Cell nuclei were stained with DAPI (Invitrogen, Life Technologies) and mounted using Mowiol solution (Sigma). As controls, uninfected and staurosporine-treated HeLa cells were also stained with DAPI as above. Images were taken using an OLYMPUS AX 70 epi-fluorescence microscope (x100 magnification), and images were visualised using Soft Imaging System Cell magnification (Olympus).

Immunofluorescence studies were performed as described previously (30 (link)). Briefly, coverslip-attached podocytes were fixed with 2% paraformaldehyde (8 min), and incubated for 60 min in blocking solution as described above. The method was similar to the flow cytometry protocol, except the permeabilization step was omitted in order to visualize surface-bound antibodies. A 60 min incubation with primary mouse antibody reactive with the insulin receptor beta subunit (IRβ subunit (mouse monoclonal, 1:300, BD, USA) was followed by a subsequent 45 min incubation with Atto550-conjugated secondary antibody (1:200, Sigma-Aldrich, Poland). Non-specific staining was controlled by replacing primary antibody with blocking solution alone. The coverslips were mounted on microscope slides using 15% Mowiol solution (Sigma-Aldrich, Poland) and examined by fluorescence microscopy (Olympus IX51), using CellSens v.1.3 imaging software (Olympus). Camera settings were identical for all samples to ensure comparable results.

Mice were perfused transcardially with ice-cold 4% paraformaldehyde (in PBS). Brains were removed and post-fixed overnight at 4 °C with the same fixative. Coronal sections were cut at 50 μm thickness for all histological analyses except for dendritic reconstructions (200 μm), using a cryostat (Leica) or a sliding microtome (Microm).
Immunofluorescence experiments were performed as described before [70 (link)]. Briefly, free-floating sections were blocked and permeabilized for one hour in a blocking buffer composed of 10% Normal Goat Serum, 0.2% Triton X-100 (Sigma) in PBS. Primary antibodies, diluted in blocking solution and added overnight at 4 °C, were as follows: rabbit anti-NEUROD2 (1:500, Abcam, #ab104430), rabbit anti-TBR1 (1:1000, Abcam, #31940), rat anti-BCL11B (1:100, Abcam, #ab18465), rabbit anti-CUX1 (1:200, Santa Cruz Biotechnology, #sc13024), mouse anti-RORβ (1:200, Perseus Proteomics, #PP-N7927-00), chicken anti-GFP (1:500, Aves, #GFP-1010). For immunocytochemistry we used mouse anti-Tuj1 (1:500, Biolegend, BLE801201). Corresponding fluorescently labeled secondary antibodies (AlexaFluor, Invitrogen) were added for 2 h in blocking solution at room temperature. Hoechst was added in PBS for 10 min, and sections were mounted on microscope slides that were coversliped using Mowiol solution (Sigma).