Sp5 dmi inverted confocal microscope

Dissociated organoid cultures were grown on growth factor-reduced Matrigel (Corning)-coated chambered coverslips and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min at room temperature. Whole cerebral organoids were fixed in 4% PFA and processed for cryosectioning (42 (link)). For immunofluorescence staining, sections or cells were immunoblocked and permeabilized in 0.25% Triton X-100 and 4% goat serum in PBS followed by overnight incubation with primary antibodies in 0.1% Triton X-100 and 4% goat serum in the following dilutions: TUJ1 (mouse; BioLegend catalog no. 801201), 1:500; PAX6 (rabbit; BioLegend catalog no. 901301), 1:100; glial fibrillary acidic protein (GFAP) (rabbit; Dako catalog no. Z0334), 1:500; doublecortin (DCX) (guinea pig; Invitrogen catalog no. A-11075), 1:500; anti-flavivirus group E antigen 4G2 (mouse; Millipore; catalog no. MAB10216), 1:200; anti-flavivirus NS5 (chicken, made in-house [14 (link)]), 1:500. Secondary antibodies consisted of Alexa Fluor 488, rabbit Alexa Fluor 594, chicken Alexa Fluor 555, and guinea pig Alexa Fluor 568 conjugates (Invitrogen) and were used in dilutions of 1:200. Images were taken with a Leica SP5 DMI inverted confocal microscope.

Cells grown in Millicell® EZ SLIDES were fixed in 4% paraformaldehyde solution for 10 min at room temperature (RT) and then incubated in blocking/permeabilizing solution (5% BSA and 0.5% Triton X-100 in PBS) for 30 min at RT. Cells then were stained with phalloidin-rhodamine (Cytoskeleton) for 30 min according to manufacturer instruction at RT. After washing three times with PBS, slides were mounted in DAPI-containing Vectashield® mounting medium (Vector Laboratoryies, Inc., Burlingames, CA). Digital images were obtained with a Leica SP5 DMI inverted confocal microscope.