Immunocytochemistry

Human myoblasts were isolated from biopsies and cultivated as described previously [19 (link)] in a growth medium consisting of 199 medium and DMEM (Invitrogen Carlsbad, CA) in a 1:4 ratio, supplemented with 20% FCS (Invitrogen), 2.5 ng/ml hepatocyte growth factor (Invitrogen), 0.1 μmol/l dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/ml gentamycin (Invitrogen). The myogenic purity of the populations was monitored by immunocytochemistry using desmin as marker. Enrichment of myogenic cells was performed using an immunomagnetic cell sorting system (MACS; Miltenyi Biotec, Paris, France) according to the manufacturer's instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Purification was checked by immunochemistry using a desmin marker. Differentiation was induced at confluence by replacing the growth medium with DMEM supplemented with 100 μg/ml transferrin, 10 μg/ml insulin and 50 μg/ml of gentamycin (Sigma-Aldrich).

For immunofluorescence experiments, different fixation methods were exploited depending on the antibodies used. For immunocytochemistry of MTs, cells were extracted for 1 min in prewarmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in MRB80 buffer (MRB80 buffer: 80 mM Pipes, 1 mM EGTA, and 4 mM MgCl₂, pH 6.8) and subsequently fixed in prewarmed 4% PFA in PBS for 10 min. For immunocytochemistry of cytochrome C, cells were fixed in prewarmed 4% PFA in PBS for 10 min. For immunocytochemistry of EB1 and detyrosinated MTs, cells were fixed in ice-cold methanol for 10 min. Samples prepared for STED imaging were extracted for 1 min in pre-warmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in MRB80 buffer and subsequently fixed in prewarmed 4% PFA (15,170; Electron Microscopy Sciences) in MRB80 buffer for 10 min. After fixation, cells were washed with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed again with PBS, and subsequently blocked for 1 h with 3% BSA in PBS. Cells were incubated with primary antibody diluted in 3% BSA in PBS for 1 h at RT, washed with PBS, and incubated with secondary antibody diluted in 3% BSA in PBS for 1 h at RT. Samples prepared for STED imaging were incubated for 2 h in primary and 2 h in secondary antibody. After washing with PBS, cells were dipped in MilliQ water, air-dried, and mounted on microscopy slides using Prolong Diamond (Molecular Probes). The following primary antibodies were used in this study: Cytochrome C (6H2.B4; BD Biosciences), EB1 (5/EB1 BD Biosciences), acetylated tubulin (6-11B-1; Sigma-Aldrich), α-tubulin (EP1332Y; Abcam), α-tubulin (B-5-1-2; Sigma-Aldrich), tyrosinated tubulin (YL1/2; Abcam), detyrosinated tubulin (AB3210; Merck), and GFP (GFP-1010; Aves Lab). DAPI (Molecular Probes) was used to visualize DNA.

ITSCs were seeded on roughened cover glasses with a density of 5 × 10⁴ Cells. Immunocytochemistry was done as previously described in Ruiz-Perera et al. [33 (link)]. Afterwards, Primary antibodies against RELA (mouse, 200,301,065, 1:5000, Rockland, PA. USA), RELB (rabbit, D7D7W, 1:500, Cell Signaling Technology, Danvers, USA), c-Rel (rabbit, 4727, 1:500, Cell Signaling Technology), vGlut-II (rabbit, 07-1402-I, 1:300, Merck Millipore, Burlington, USA), NF200 (mouse, SAB3200747, 1:200, Sigma Aldrich), MAP2 (mouse, sC-390,543, 1:500, Santa Cruz, Dallas, USA) and synaptophysin (rabbit, ab32127, 1:500, Abcam, Cambridge, UK) were incubated for 1 h at RT. Secondary fluorochrome-conjugated antibodies (Alexa 488 anti-rabbit, Alexa 555 anti-mouse, Alexa 555 anti-rabbit, 1:300, Life Technologies, Germany) were incubated for 1 h at RT under the exclusion of light, followed by DAPI nuclear counterstaining. Fluorescence microscopy was performed using the confocal laser scanning microscope LSM 780 (Carl Zeiss, Jena, Germany). Randomly placed pictures were analyzed using ImageJ [34 ]. Nuclear fluorescence intensity was determined using the “measure” function in ImageJ on the nuclei of the cells.

The immunostaining pattern of claudin-5, occludin, ZO-1 and P-gp was studied in primary BECs isolated from WT and APOB-100 transgenic mice. Cells were fixed with ice-cold acetone-methanol for 2 min, then non-specific binding was blocked with 1% BSA in PBS at room temperature during 1 h. Cells were incubated with primary antibodies shown in Additional file 1: Table S2 overnight at 4 °C, which was followed by a 1 h incubation with the corresponding secondary antibodies (A594-conjugated donkey anti-rabbit, A488-conjugated goat anti-mouse (Thermo Fisher Scientific, MA, USA) and Cy3-labeled sheep anti-rabbit, as shown in Additional file 1: Table S2). Cellular nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientific) at a concentration of 1 µg/ml. The samples were mounted (Fluoromount-G; Southern Biotech, AL, USA), then examined using a Spinning Disk Confocal Microscope (Zeiss, Germany).
Astroglia and microglia were immunolabeled to visualize GFAP, S100B, AQP4 and Iba-1 expression, respectively. Glial cells were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific binding of antibodies was blocked with 3% BSA in the case of S100B + AQP4 co-staining, and with 2% normal horse serum and 5% normal goat serum for Iba-1 + GFAP co-labeling. Glial cells were incubated with primary antibodies (Additional file 1: Table S2) overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for 1 h (A488-labeled donkey anti-goat (Thermo Fisher Scientific), DyLight 549-conjugated goat anti-mouse (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK), A594-conjugated donkey anti-rabbit (Thermo Fisher Scientific), as shown in Additional file 1: Table S2. Hoechst dye 33,342 was used for nuclear staining at a concentration of 1 µg/ml. After mounting the samples (Fluoromount-G; Southern Biotech), the immunoreactivity was examined using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Germany).
Isolated brain microvessels were fixed with 3% paraformaldehyde immediately after cytokine treatment, and the expression pattern of key BBB proteins claudin-5, occludin, ZO-1, P-gp and AQP4 was analyzed using immunocytochemistry. Microvessels were permeabilized and non-specific binding was blocked with 0.2% Triton X-100 and 2% normal serum in PBS for 10 min. Then microvessels were incubated overnight at 4 °C with primary antibodies at dilutions shown in Additional file 1: Table S2. The next day, microvessels were incubated for 50 min (Additional file 1: Table S2) with the corresponding secondary antibodies, i.e. A594-conjugated donkey anti-rabbit and A488-conjugated goat anti-mouse (Thermo Fisher Scientific), as shown in Additional file 1: Table S2. Cellular nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) at a concentration of 1 µg/ml. Between incubations microvessels were washed three times with PBS. The staining patterns were examined with a Spinning Disk Confocal Microscope (Zeiss, Germany).