Mab1435

Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature (RT). The samples were blocked with 5% normal goat serum in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT. They were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, the samples were placed in mounting medium with 4',6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX, USA), NANOG (ReproCell), Tra1-60 (MAB4360; Sigma-Aldrich), SSEA4 (MAB1435; R&D Systems), SSEA1 (MAB2155; R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin, USA), α-smooth muscle actin (α-SMA; A2547; Sigma-Aldrich), and SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies except for the anti-TUJ1 antibody (1:300) were used at a 1:150 dilution in 5% normal goat serum.

Nanoparticle variants at a final concentration of 25 μg mL⁻¹ were applied to well plates in duplicate, to allow for immuno-histochemical identification of multiple cell types and nanoparticle localization therein, and cultures were incubated for a further 24 hours at 37 °C in 5% CO₂, with the inclusion of 20% FBS in the culture media as described in Section 2.7. Experiments were repeated three times using cultures derived from separate litters of rat pups. Immunohistochemistry was conducted in accordance with established procedures^{5 (link)} using primary antibodies recognizing markers as follow: microglial activation markers Iba1 (1:500; Abcam, goat Ab5076) and ED1 (1:500; Merck Millipore, mouse MAB1435); oligodendroglial indicator oligodendrocyte transcription factor 2 (Olig2; 1:500; R&D Systems, goat AF2418); astrocyte indicator glial fibrillary acidic protein GFAP (1:500; Abcam, rabbit AB33922); and neuronal indicator β-III tubulin (1:500; Merck Millipore, mouse MAB1637). Antibodies were diluted in PBS containing 0.2% Triton™ X-100 and 5% normal donkey serum. Secondary antibodies were Alexa Fluor 488 or 555 (1:400; Thermo Fisher Scientific™), together with Hoechst 3342 (1:1000; Thermo Fisher Scientific™) diluted in 1× PBS containing 0.2% Triton™ X-100. Finally, the sections were mounted on glass slides with cover slips using Fluoromount-G (Thermo Fisher Scientific™). The slides were viewed using Nikon Eclipse Ti-inverted microscope. Four randomly sampled, non-overlapping Z-stack images were taken per well at 20× magnification. A series of 13 optical images were taken at 0.5 μm increments along the z-axis, and deconvoluted using Nikon Elements NT software. This was followed by the assessment of consistently sized (293.703 × 293.703 μm), non-overlapping regions of interests (ROIs) in the confocal images in order to determine the proportion of distinct cell populations that exhibit Cy5 fluorescence from the uptake of NP and Tf-NP. All image analyses were performed on Fiji image processing software (National Institutes of Health). In conjunction with representative confocal images obtained using all of the above-mentioned markers, total numbers of Iba1+ resident reactive microglia and ED1+ activated microglia/macrophages with and without nanoparticles were counted within a ROI in a 40× image and expressed as the mean number of cells per mm². Quantification was confined to microglia/macrophages due to the prevalence of nanoparticle variants within these cell populations, and the biological relevance of immune cell engulfment of nanoparticles.