Fluoroshield mounting medium

Embedded tissues were cut into 5 μm thick and placed in electrocharged slides; then, histological sections were deparaffinized and gradually rehydrated. For histological examination, samples were stained using the standard H&E method. For IF, after sample rehydration, antigen was retrieved according to the Rodent Decloaker kit (BioCare Medical. CA, USA). Then, they were blocked with 1% BSA for 2 h and incubated overnight at 4 °C with the following primary antibodies: Annexin A1 (ANXA1, 1:50; NBP1-90162; RRID: AB_11018791), Annexin A2 (ANXA2, 1:50; NBP1-31310; RRID: AB_2242775), and Annexin A5 (ANXA5, 1:50; NBP2-38248); all antibodies were from NOVUS Biologicals. CO, USA. Then, tissues were incubated for 1 h at room temperature in the dark with an Alexa Fluor 488 anti-rabbit antibody (1:300, ab150077; RRID: AB_2630356, Abcam, MA, USA). Next, nuclear DNA was stained with DAPI (1:1000; 62.247; Thermo Scientific, IL, USA) for 5 min at room temperature. Finally, tissues were fixed with Fluoroshield mounting medium (Thermo Scientific, IL, USA), and IF images were captured with ZEISS Axio-A1 Microscopy (Carl-Zeiss, Oberkochen, Germany).

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).