Tcs sp8 microsystems confocal microscope

LN229 and T98G cells were seeded onto LabTek II, CC2-treated 4-well chamber slides (Nalge Nunc) at a seeding density of 3 × 10⁴ cells/well. Cells were allowed to adhere overnight at 37 °C with 5% CO₂. Medium was replaced with OptiMEM (Invitrogen, Thermo Fisher Scientific, UK) 2 h before cells were transfected for 4 h with 0.5 μg/μL pEGFP-H-Ras using the RALA peptide delivery system at N:P 10 [17 (link)]. After 24 h, cells were treated with RALA/ALN NPs at mass ratio 10:1 for 6 h and then incubated for a further 72 h prior to fixation with 4% formaldehyde. Slides were sealed with a coverslip and Fluoroshield mounting medium containing DAPI nuclear stain (Thermo Fisher Scientific, UK). Slides were imaged using a TCS SP8-Leica Microsystems confocal microscope (Leica, UK) with a 63x oil objective lens, 1024 × 1024 frame and 400 Hz scanning speed. Images were analysed using LAS AF Lite Software (Leica, UK) and Fiji ImageJ (National Institute of Health, USA).

Cells were seeded onto 24-well culture multi-dish plates on 13 mm coverslips (Agar Scientific, UK) at 3 × 10⁴ cells/well and allowed to adhere overnight at 37 °C with 5% CO₂. Medium was replaced with OptiMEM (Invitrogen, Thermo Fisher Scientific, UK) 2 h prior to treatment. Cells were treated with RALA/AF647-RIS NPs at a mass ratio 10:1 containing 0.1 μg AF647-RIS. NPs were prepared as described in section “Formulation of RALA/ALN NPs”. in a total volume of 50 μL and stored under dark conditions. Cells were incubated with NPs for 2 h, before media was removed. Following treatment, cells were washed three times with warmed PBS, fixed with 4% formaldehyde and permeabilised using 0.1% Triton X -100 (Sigma-Aldrich, UK). The cytoskeleton was stained with AlexaFluor®488-phalloidin (Invitrogen, Thermo Fisher Scientific, UK) for 15 min at RT. Following staining, cells were washed with warmed PBS and mounted on microscope slides (Agar Scientific, UK) with Fluoroshield mounting medium containing DAPI nuclear stain (Invitrogen, Thermo Fisher Scientific, UK). Slides were imaged using a TCS SP8-Leica Microsystems confocal microscope (Leica, UK). Images were analysed using LAS AF Lite Software (Leica, UK).

N2a cells on coverslips were fixed in 4% paraformaldehyde for 10 mins and washed with PBS three times. Mice brain tissues were fixed in 4% paraformaldehyde for 8 h, Sucrose gradient dehydration, frozen, and sliced into 30mm‐thick slices (8mm‐thick for embryonic mice brain) with a Leica CM1950 sectioning cryostat. Nuclei were stained with DAPI. The primary antibodies used in this study included KDM7A (cat. No. A14692; 1:100, ABclonal), Tuj‐1 (cat. No. MAB1195; 1:100, R&D), GFAP (cat. No. G3893; 1:100, MilliporeSigma), CX3CR1 (cat. No. AF5825; 1:100, R&D), c‐Fos (cat. No. 2250; 1:100, CST). Coverslips with adult mice brain slices were observed using a TCS SP8‐Leica Microsystems confocal microscope (Leica, UK) with a 63×oil objective lens and subsequently analyzed using LAS AF Lite Software (Leica, UK). Coverslips with embryonic mice brain slices were observed using Olympus Slideview VS200 and subsequently analyzed using Olyvia‐3.3. Cells with immunoreaction were defined as “positive” cells. ^[44] Positive cells were counted by a trained observer who was blind to the treatment of the animals. Three sections from each sample were analyzed and the mean percentage of each sample was calculated. Numbers of samples analyzed in different experiments were stated in Figure legends.