Immunofluorescence Imaging of Cultured Neurons

aiFACS-sorted neurons were plated on ornithine-coated glass coverslips (35-mm diameter) and cultivated in complete medium: neurobasal (Invitrogen) supplemented with B-27 (Invitrogen) and GlutaMAX (Invitrogen) as previously described (Abekhoukh et al. 2017 (link); Maurin et al. 2019 (link)). Neurons were fixed, and immunofluorescence was performed with microtubule associated protein 2 (MAP2) antichicken polyclonal antibody (BioLegend 822501) detected with a secondary goat antichicken Alexa 594 (Invitrogen A32759) 6 d after the selection, as previously described (Drozd et al. 2019 (link)). Fluorescent images were taken using a wide-field upright fluorescence microscope (Axioplan2, Carl Zeiss), with an ORCA ER CCD camera (Hamamatsu), through a rhodamine filter set (BP565/30; LP585; BP620/60) and a PlanApoChormat 63×/1.4 DIC oil immersion objective (pixel size: 100 nm).
Microglia were labeled as previously described (Cazareth et al. 2014 (link)) using the following antibodies: BV510 anti-mouse CD45-clone 30-F11 (BD Biosciences 563891) and AlexaFluor700 anti-mouse CD11b-clone M1/70 (Sony Biotechnology 1106110).

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Castagnola S., Cazareth J., Lebrigand K., Jarjat M., Magnone V., Delhaye S., Brau F., Bardoni B, & Maurin T. (2020). Agonist-induced functional analysis and cell sorting associated with single-cell transcriptomics characterizes cell subtypes in normal and pathological brain. Genome Research, 30(11), 1633-1642.

Publication 2020

neurobasal GlutaMAX goat antichicken Alexa 594 Axioplan2 ORCA ER CCD camera

Alexa 594 Antibodies Antibody Cd11b Clone Fluorescence microscope Goat Immersion Immunofluorescence Microglia Microtubule associated protein 2 Mouse Neurons Orca Ornithine Rhodamine

Corresponding Organization :

Other organizations : Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d'Azur, Centre National de la Recherche Scientifique, Inserm

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Variable analysis

independent variables

Neuron culture type (aiFACS-sorted neurons)

dependent variables

Neuron morphology (assessed via immunofluorescence of MAP2)

control variables

Neuron plating on ornithine-coated glass coverslips
Neuron culture medium (neurobasal, B-27, GlutaMAX)
Neuron culture duration (6 days)

positive controls

Immunofluorescence staining for microtubule associated protein 2 (MAP2)

negative controls

Not specified

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