Immunofluorescence Staining of Cerebral Organoids

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.

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Janssens S., Schotsaert M., Karnik R., Balasubramaniam V., Dejosez M., Meissner A., García-Sastre A, & Zwaka T.P. (2018). Zika Virus Alters DNA Methylation of Neural Genes in an Organoid Model of the Developing Human Brain. mSystems, 3(1), e00219-17.

Publication 2018

Matrigel GFAP SP5 DMI inverted confocal microscope

Alexa 568 Alexa 594 Alexa fluor 488 Alexa fluor 555 Antibodies Cells Chicken Confocal microscope Dilutions E antigen Flavivirus Glial fibrillary acidic protein Goat Growth factor Guinea pig Immunofluorescence Matrigel Mouse Organoids Paraformaldehyde Phosphate Rabbit Saline Serum Triton x 100

Corresponding Organization :

Other organizations : New York Stem Cell Foundation, Icahn School of Medicine at Mount Sinai, Harvard University, Harvard Stem Cell Institute, Broad Institute

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Variable analysis

independent variables

Growth factor-reduced Matrigel (Corning)-coated chambered coverslips
Fixation with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min at room temperature

dependent variables

Immunofluorescence staining of TUJ1, PAX6, GFAP, DCX, anti-flavivirus group E antigen 4G2, and anti-flavivirus NS5

control variables

Incubation conditions (overnight incubation with primary antibodies in 0.1% Triton X-100 and 4% goat serum)
Secondary antibody dilutions (1:200)

positive controls

No positive controls specified

negative controls

No negative controls specified

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