Goat anti rabbit igg conjugated with alexa fluor 594

Tissues of MCAO+ SKP-SCs group were directly observed under a fluorescence microscope (ZEISS, Germany) to detect survival and migration of GFP-labeled SKP-SCs in the rat brain at 6 h, 12 h on Day 0, Days 1, 4, 7, 14 and 28 after MCAO. To further observe the health of neurons and glial cells, as well as neuronal dendritic processes and axonal growth, additional sets of slides were stained with antibodies against different neuronal and glial cell markers, including NeuN, GFAP, MAP2, and GAP43. After temperature equilibration and drying, sections mounted on slides were washed with PBS for 10 min, then blocked with 0.1% Triton x-100 for 2 h, before being incubated overnight at 4°C with the following antibodies: rabbit anti-NeuN antibody (1:500, Abcam, USA) and rabbit anti-GFAP antibody (1:500, Abcam, USA), mouse anti-MAP2 antibody (1:500, Abcam, USA), and rabbit anti-GAP43 antibody. Indirect fluorescence by incubating sections at room temperature for 2 h with goat-anti-rabbit IgG conjugated with Alexa Fluor 594 or goat-anti-mouse-IgG conjugated with Alexa Fluor 488 (1:1000, Life Technologies) was used for detection. Sections were further incubated with Hoechst for 10 min before being coverslipped for microscopic observation.

SK-N-SH and RD cells (∼80% confluent) in a 96-well plate were infected with EV-D68 at an MOI of 2. Mock-infected cells were included as a negative control. EV-D68-infected SK-N-SH and RD cells were incubated at 37°C and 33°C in 5% CO₂, respectively. After 8 hpi, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, and permeabilized with 70% ethanol. Cells were first incubated with 5% bovine serum albumin (BSA; Aurion, Wageningen, The Netherlands) in PBS for 30 min before incubation with rabbit anti-EV-D68 VP1 (20 μg/ml; GeneTex, Irvine, CA, USA) for 1 h. Cells were washed twice with PBS and incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 594 (10 μg/ml; Life Technologies, Inc., The Netherlands) in PBS with 0.1% BSA (Aurion) for 1 h. Cells were washed 3 times with PBS and mounted with ProLong Diamond Antifade with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies) to visualize nuclei. Each experiment included negative and omission controls. EV-D68 VP1-positive cells were identified using a Zeiss LSM 700 laser scanning microscope. All images were processed using Zen 2010 software. Per sample, 3 high-power fields were photographed, and the number of infected cells was calculated by counting virus-infected/uninfected cells in 3 randomly chosen panels. All experiments were performed in triplicate.

Goat anti‐guinea pig (Cat# A11073) or mouse IgG conjugated with Alexa Fluor 488 (Cat# A11029) and goat anti‐rabbit IgG conjugated with Alexa Fluor 594 (Cat# A11037) were purchased from ThermoFisher Sci, Waltham, MA.