Donkey anti rabbit 594

BJ senescent cells were reverse transfected with control siRNA and p53 siRNA and seeded on LabTek 8-well chambers slides (Thermo Fisher Scientific) at a density of 1.4 × 10⁴ cells per well. One day after, the cells were exposed to 500 vge/cell of AF488-labelled PsVs. 48 h after infection and 72 h after transfection, cells were fixed in 3.7% paraformaldehyde for 15 min, incubated for 1 h with 10% goat serum and then stained using antibodies for early endosomes (rabbit anti-EEA1, C45B10, Cell Signaling Technology) and lysosomal markers (mouse anti-LAMP1, D4O1S, Cell Signaling Technology). Donkey anti-rabbit 594 and goat anti-mouse rhodamine were used as secondary antibodies (Molecular Probes). Slides were visualised using a Zeiss Axiovert 100 M microscope attached to an LSM 510 confocal unit.

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min, followed by a 5-min permeabilization step with 100% methanol, washed twice with PBS for 5 min per wash. Next, the cells were blocked with 0.1% Tween (Sigma-Aldrich) and 10% normal donkey serum blocking solution (Sigma-Aldrich) in PBS at room temperature. Cells were incubated overnight at 4°C with primary antibodies; dilution of 1:100 rabbit anti-αSMA (Abcam #ab5694), 1:100 rabbit anti-Nanog (Abcam #ab21624), and 1:250 mouse anti-smMHC11 (Abcam #ab683). The cells were then incubated for 1 h at room temperature with secondary antibodies donkey anti-mouse 488 (Thermo Fisher Scientific #a21202) and donkey anti-rabbit 594 (Thermo Fisher Scientific #A21207) at 1:200 dilution. After rinsing, cells were incubated with a drop of NucBlue Fixed Cell Ready Probes Reagent (Thermo Fisher Scientific #R37606) in PBS for 5 min. Cells were stored at 4°C in the dark. Examination was done using the confocal microscope A1 (Nikon Instruments Inc), and all images were processed with Elements 3.20 software (Nikon Instruments Inc).

Brain tissues of patients with ICH and ICH mice were fixed in 4% paraformaldehyde and embedded in paraffin. Brain sections (5 µm thick) were made and rehydrated in a series of ethanol dilutions. Brain sections were permeabilized and incubated in blocking solution (5% goat or donkey serum in PBS solution), followed by incubation with primary antibodies: goat anti-Iba1 (Wako, Richmond, VA, USA) and rabbit anti-PBR (Abcam, Cambridge, MA, USA) at 4°C overnight. After triple washings with PBS, the slices were incubated with secondary antibodies: donkey anti-rabbit 594 (Thermo Fisher Scientific) and donkey anti-goat 488 (Thermo Fisher Scientific). After triple washings with PBS, the brain sections were stained with DAPI (Abcam). For TUNEL staining, the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling kit (Roche, Indianapolis, IN, USA) was used according to the manufacturer’s protocol. Images were captured with a microscope (Model BX-61; Olympus, Center Valley, PA, USA), and data were analyzed with Image J (NIH). For cell counting, positively stained cells were counted in 3 comparable, randomly selected microscopic fields. The numbers of cells from 9 locations per mouse (3 fields per section × 3 sections per mouse) were averaged and expressed as positive cells per field.

Immunofluorescence staining was done 24 or 48 h after transfection or electroporation. Cells were rinsed once with 1x PBS and fixed with 4% PFA at room temperature for 15 min. Fixed cells or brain slices were rinsed with 1x PBS, incubated with blocking buffer (1x PBS with 0.3% Triton-X-100 and 5% donkey serum) at room temperature for 30 min, and further incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. After three brief rinses in 1x PBS, slides were incubated for 1 h at room temperature with fluorophore-conjugated secondary antibodies (Donkey anti-chicken 488 (Jackson ImmunoReseach, 703-546-155), donkey anti-mouse 488 (Thermo Scientific, A21202), donkey anti-rabbit 594 (Thermo Scientific, A21207), donkey anti-rat 647 (Thermo Scientific, A48272); 1:1000 dilution for all). Slides were scanned with a Leica SP5 inverted confocal microscope. The following primary antibodies were used: anti-HA (Millipore 3F10 clone, rat, 1:1000), anti-GFP (Abcam ab13970, chick, 1:2000), anti-PUM2 (Bethyl A300-202A, rabbit, 1:1000), anti-CD24-PE (BioLegend 138504, rat, 1:1000), anti-CD133-APC (BioLegend 141208, rat, 1:1000), anti-PAX6 (Covance PRB-278P, rabbit, 1:500), anti-APP (BioLegend 802803, mouse, 1:1000).