Donkey anti rabbit af488

Immunofluorescence staining was performed to demonstrate the association of NS1 with CD133 and CD34 in UCB. After blocking in 1% BSA, the cells were incubated with primary antibodies CD34-FITC and CD133-PE (BD Biosciences, Franklin Lakes, NJ, USA). Then, the cells were fixed with 4% paraformaldehyde and attached to slides using a cytospin (Thermo Scientific, MA, USA) at 400 rpm for 8 min. The monolayer was washed twice in PBS and permeabilized (0.3% Triton X-100 with 1% BSA in PBS) and finally washed twice again in PBS. The cells were labeled using NS-1 rabbit polyclonal antibody (Gentex, Zeeland, MI, USA) in a permeabilization buffer at 4 °C for 2 h and washed twice again in PBS. The cells were further incubated with AF-488-donkey anti-rabbit (Invitrogen, Carlsbad, CA, USA) and AF568-goat anti-rabbit (Life Technologies, Carlsbad, CA, USA) secondary antibodies for 1–2 h in the dark at room temperature and covered with the antifade reagent DAPI. Fluorescent images were captured in an Inverted Confocal Microscope FV-1000 (Olympus, Shinjuku City, TYO, Japan).

Primary antibodies used in this study include ARL13b (Proteintech catalog 17711-I-AP), acetylated tubulin (Sigma catalog T6793), IFT88 (Thermo Fisher Scientific catalog PA5-18467), Inversin (Proteintech catalog 10585-I-AP), Dynein (Thermo Fisher Scientific catalog MA1-070), gTubulin (GeneTex catalog GTX113286), Alk1 (Abcam catalog ab51870), KLF4 (Proteintech catalog 11880-I-AP), HO-1 (BD, catalog 566391), NRF2 (BioLegend, catalog 939202), and bactin (Sigma catalog A5441 and Cell Signaling Technology catalog 4970P). Secondary antibodies used are goat anti-mouse PECy7 (BioLegend), donkey anti-rabbit PE (Thermo Fisher Scientific), donkey anti-goat AF657 (Thermo Fisher Scientific), donkey anti-rabbit BV421 (BioLegend), and donkey anti-rabbit AF488 (Thermo Fisher Scientific).

Astrocytes isolated from E15 and cultured on poly-D-lysine-coated coverslips were stained with 5 μM MitoTracker DeepRed to label mitochondria. Alternatively, cells were transfected with ER-dsRed expression vector 1 day prior to fixation to label ER. Cells were fixed with 4% paraformaldehyde for 15 min and were then permeabilized by ice-cold methanol for 5 min, followed by PBS rinse for 10 min. The cells were then blocked with 5% bovine serum albumin in PBS with 0.3% TritonX-100 for 1 h, followed by primary antibody [rabbit-anti-HSP60 (CST), rabbit-anti-LC3B (CST), rabbit-anti-LAMP1 (CST), rabbit-anti-ACSL4 (Abcam), mouse-anti-STAT3 (CST), both diluted at 1:200 in the blocking buffer] incubation at 4°C overnight. Then, the cells were incubated in appropriate secondary antibodies [donkey-anti-rabbit-AF488, donkey-anti-rabbit-647, donkey-anti-mouse-488, donkey-anti-mouse-AF568 or donkey-anti-mouse-AF647 (Thermo), all diluted at 1:200 in the blocking buffer] for 1 h at room temperature. After washing three times in TBST, the cells on the coverslip was mounted using Prolong Gold with DAPI (Thermo) and subjected to confocal microscopy observation using the Zeiss LSM880 with Airyscan system. Images were captured using a 63x/1.4NA oil immersion objective. The colocalization analysis was performed with Fiji software using the Coloc 2 plugin.