Neurospheres were plated on PLOF-coated glass coverslips and allowed to attach for 2 days, fixed in 4% (w/v) paraformaldehyde (PFA) + 4% (w/v) sucrose in PBS for 30 min and processed for immunostaining as previously described41 (link). Primary and secondary antibodies were used as follows: mouse anti-βIII-tubulin (1:200; Millipore Darmstadt, Germany, MAB1637); mouse anti-GFP (1:200; Sigma-Aldrich, St Louis, MO, USA, G6539); rabbit anti-TH (1:100; Santa Cruz Biotechnology, Dallas, TX, USA, sc-14007); rabbit anti-GFAP (1:200; Millipore, AB5804); rabbit anti-CAR (1:200; gift from Joseph Zabner), mouse anti-CAR (1:200; Millipore, 05-644); AlexaFluor® 488 goat anti-mouse IgG (1:500; Life Technologies, A11001) and AlexaFluor® 594 goat anti-rabbit IgG (1:500; Life Technologies, A11012). Cell nuclei were counterstained with DAPI or TO-PRO-3 (Life Technologies). Samples were visualized using point scan confocal microscopy (SP5, Leica, Wetzlar, Germany). Merge between channels and maximum z-projections, as well as linear brightness and contrast adjustments of the images, were performed using the ImageJ software.
Protocol detail
Immunofluorescence Staining of Neurospheres
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mouse anti-βIII-tubulin
mouse anti-GFP
rabbit anti-TH
rabbit anti-GFAP
AlexaFluor® 488 goat anti-mouse IgG
AlexaFluor® 594 goat anti-rabbit IgG
TO-PRO-3
Anti igg
Antibodies
Cell nuclei
Confocal microscopy
Dapi
Gfap
Goat
Mouse
Paraformaldehyde
Rabbit
Scan
Sucrose
To pro 3
Tubulin
Corresponding Organization :
Other organizations : Instituto de Biologia Experimental e Tecnológica, The Francis Crick Institute, The Honourable Society of Lincoln's Inn, Sapienza University of Rome, Institut de Génétique Moléculaire de Montpellier, Sobell House, University College London, Universidade Nova de Lisboa
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Variable analysis
independent variables
- Culturing of neurospheres on PLOF-coated glass coverslips
- Expression of βIII-tubulin, GFP, TH, GFAP, and CAR proteins in neurosphere cells
- Neurosphere culture and attachment duration (2 days)
- Fixation in 4% (w/v) paraformaldehyde (PFA) + 4% (w/v) sucrose in PBS for 30 min
- Antibody concentrations (1:200 for primary antibodies, 1:500 for secondary antibodies)
- Counterstaining of cell nuclei with DAPI or TO-PRO-3
- Visualization using point scan confocal microscopy
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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