Glycergel mounting medium

Labelled tissue sections were observed on an Olympus BX61 microscope (Olympus, Tokyo, Japan), and image processing was performed using Xcellence Rt software version 1.18 (Olympus).
Fixed cells were permeabilized with PBS containing 0.1% Triton X-100 and 3% BSA for 10 min. Cells were subsequently stained with 2 mg/mL Hoechst (Thermo Fisher Scientific) to visualize nuclei. To visualize the actin cytoskeleton, some samples were also stained with phalloidin-rhodamine (Merck). After labeling, samples were mounted on glass slides using Glycergel Mounting Medium (Agilent Technologies, Santa Clara, CA, USA). Stained cells were imaged with a Nikon Eclipse Ti-E system equipped with a 100× oil immersion objective and a Zyla sCMOS camera (Oxford Instruments, Abingdon, UK), controlled by the NIS-Elements software from Nikon. The fluorescence illumination system was a CoolLED pE-300white (CoolLED, Andover, UK). Fluorescence filter sets for DAPI, GFP/FITC, and TexasRed were used to detect Hoechst, EGFP, and rhodamine, respectively. All images contained 9 z-stacks with a 500 nm step-size in z.

After PCL- or PLLA-NP exposure, cells grown in 0.1% gelatin-coated 96-well plates or on coverslips were fixed with cold 4% paraformaldehyde for 20 min at room temperature. Following two washing steps with Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies, UK), cells were blocked with 10% horse serum in 0.4% Triton-PBS for 1–2 h at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4 °C (Table 1). After washing four times with PBS, except for Acti-stain 488 phalloidin, the corresponding secondary antibodies were applied and left for 2 h before cells were washed again four times with PBS. Thereafter, coverslips were mounted on glass slides using Glycergel Mounting Medium (Dako, Denmark/USA). The samples were examined and images were obtained using a Zeiss Axio Imager Z1 coupled with an Apotome 1 (Carl Zeiss Vision Swiss AG, Feldbach, Switzerland).
To further study uptake of PCL-, PLLA- and Au-NPs, rBCEC4 cells were seeded at 150,000 cells (24- well plate), coated with 0.1% gelatin and were incubated in cell culture medium at 37 °C and 5% CO₂ until full confluence was reached. Cells were then exposed to PLLA-NPs for 2 h, to PCL-NPs for 24 h and Au-NPs for 2 and 24 h. Subsequently, TEM was performed as previously described [11 (link)].

Mice were perfused transcardially with 4% paraformaldehyde (PFA) in PBS, and brains were dissected. Brainstems were fixed in PFA solution for 2 h, then equilibrated in a 30% sucrose solution in PBS overnight. Brains were cryosectioned at 16 μm in the coronal plane at the level of the auditory brainstem nuclei. Sections were mounted on chrome-alum glass slides in a 1-in-4 (P1) or 1-in-6 (P6 and P14) series. Mounted sections were then surrounded with a Pap Pen hydrophobic barrier and rinsed in PBS for 10 min. For antigen retrieval, sections were incubated with 0.1% SDS in PBS solution for 5 min. Slides were rinsed three times in PBS, then incubated with either normal goat blocking solution (4% normal goat serum, 0.1% Triton X-100 in PBS), or bovine serum albumin blocking solution (4% bovine serum albumin, 0.4% Triton X-100 in PBS) for 1 h in a humid chamber at room temperature. Bovine serum albumin blocking solution was used for synaptic marker immunolabeling, and normal goat blocking solution was used for immunolabeling of glial cells. Primary antibodies (see below) were applied, and slides were incubated overnight in a humid chamber. Slides were washed in PBS and incubated at room temperature for 1 h with appropriate Alexa Fluor (Invitrogen) secondary antibodies diluted 1:500. Slides were rinsed three times in PBS and coverslipped with Glycergel mounting medium (Dako C0563).

Tissue sections were stained with recombinant CLEC10A as described previously [28 (link)]. Briefly, sections were deparaffinized and antigen retrieval was achieved by boiling in 0.1 M sodium citrate buffer (pH 5.0). Slides were blocked by 3% hydrogen peroxide and with TSM buffer in the presence of 0.2% BSA, 10% fetal calf serum and 0.3% Triton X-100. Tissue sections were incubated with complexed CLEC10A consisting of myc-tagged CLEC10A, streptavidin-horseradish peroxidase (HRP) conjugate (Thermo Fisher Scientific), and the biotinylated anti cmyc antibody 9E10 (Santa Cruz Biotechnology). After washing (3× for 5 min each) in TSM buffer, staining was performed with 3, 3′-diaminobenzidine chromogen solution (DAB; Dako) Nuclei were counterstained by hematoxylin. Stained tissue sections were coverslipped applying Glycergel Mounting Medium (Dako). Images were acquired using an Olympus BX43 microscope.

Cell cultures were immunostained as previously described (Madeira, Boia, et al., 2016), using the antibodies listed in Table 1. Human organotypic retinal cultures, following fixation with 4% PFA with 4% sucrose solution for 10 min, were blocked with 1% Triton X‐100, 1% BSA and 10% goat serum, in PBS, for 60 min. Primary antibodies (Table 1) were incubated overnight at 4°C. Retinal explants were washed with PBS and incubated with the secondary antibodies (Table 1) overnight at 4°C. Cultures were washed with PBS and incubated with 4′,6‐diamidine‐2′‐phenylindole dihydrochloride (DAPI; Invitrogen, Carlsbad, CA; 1:2000) for 10 min. The preparations were washed with PBS and mounted with Glycergel mounting medium (DAKO, Agilent, Santa Clara, CA). In all experiments, cells were observed with a confocal microscope (LSM 710, Zeiss, Oberkochen, DE), and from each condition, five random fields were acquired (20× objective), unless otherwise mentioned.