The cells were washed with DPBS and fixed at room temperature for 15 min with 4% paraformaldehyde (Merck Millipore), and washed with DPBS. The fixed cells were blocked for a minimum of 30 min in a blocking solution consisting of KPBS with 0.25% triton-X100 (Fisher Scientific) and 5% donkey serum. The primary antibody (rabbit anti-FOXG1, 1:50 dilution, Abcam, RRID: AB_732415; mouse anti-OCT3/4, 1:500, Santa Cruz Biotechnology, RRID: AB_628051; mouse anti-MAP2, 1:1000, Abcam, RRID: AB_2138153; rabbit anti-PAX6, 1:1000, Biolegend, RRID: AB_2565003; mouse anti-NEUN, 1:1000 dilution, Millipore, RRID: AB_2298772; rabbit anti-TBR1, 1:500, Abcam, RRID: AB_2200219) was added and incubated at room temperature overnight. On the following day, the cells were washed with KPBS and blocked for at least 10 min in donkey serum blocking solution. The secondary antibody (donkey anti-rabbit Cy3, donkey anti-rabbit Cy2, donkey anti-mouse Cy2; 1:200; Jackson Lab) was added with the nuclear stain DAPI (1:1000; Sigma-Aldrich) and incubated at room temperature for approximately 1 h, followed by 2–3 rinses with KPBS. The immunocytochemically labelled cells were then visualized with a Leica microscope (model DMI6000 B), and images were cropped and adjusted in Adobe Photoshop CC 2015.
Rabbit anti foxg1
Manufactured by Abcam
Sourced in United Kingdom, United States
Rabbit anti-FOXG1 is a primary antibody that specifically binds to the FOXG1 protein. FOXG1 is a transcription factor that plays a role in brain development and neural stem cell regulation.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Dapi nuclear stain, by Merck Group (1 mentions)
Cy2 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Donkey anti mouse cy2, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Mouse anti oct3 4, by Santa Cruz Biotechnology (1 mentions)
Mouse anti map2, by Abcam (1 mentions)
Rabbit anti pax6, by BioLegend (1 mentions)
Rabbit anti tbr1, by Abcam (1 mentions)
Dmi6000 b, by Adobe (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Mouse anti gapdh, by GeneTex (1 mentions)
Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse anti β 3 tubulin, by Merck Group (1 mentions)
Rabbit anti γh2ax, by Cell Signaling Technology (1 mentions)
Mouse anti nestin, by Merck Group (1 mentions)
Mouse anti map2, by Merck Group (1 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
7 protocols using rabbit anti foxg1
1
Immunocytochemical Staining Protocol
2
Foxg1 Expression in Neocortex Development
3
Immunostaining Protocol for hNPCs
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hNPCs were fixed in formaldehyde 3.7% for 15 min at RT and permeabilized in 0.2% Triton X-100 for 15 min Primary antibodies were incubated overnight in 2% BSA at 4 °C, following 40 min of 2% BSA blockage. After washing with PBS, secondary antibodies were incubated for 1 h at RT in the dark. Cells were washed three times with PBS and nuclei were stained with DAPI. Coverslips were mounted on slides using Aqua-Poly Mount (Polysciences) whereas cells on the 384 well plates were covered with glycerol and sealed with AlumaSeal CS (Excel Scientific) for image acquisition in confocal microscopy (Leica) and Operetta (Perkin Elmer), respectively. Primary antibodies used: mouse anti-MAP2 (Sigma-Aldrich), mouse anti-Ki-67 (BD Biosciences), rabbit anti-PAX6 (Santa Cruz Biotechnology), rabbit anti-GFAP (Dako), rabbit anti-γ-H2AX (Cell Signaling Technology), rabbit anti-FOXG1 (Abcam), rabbit anti-DYRK1A (Sigma-Aldrich), rabbit anti-SOX2 (Millipore), mouse anti-β-tubulin III (Millipore), mouse anti-nestin (Millipore), rabbit anti-TBR2 (Millipore). Secondary antibodies used: goat anti-mouse Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific). Data are expressed as relative protein expression in comparison with basal protein expression in control with vehicle (DMSO).
4
Western Blot Analysis of Cell Signaling Pathways
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Western blotting was carried out according to the Bio-Rad general protocol (BIO-RAD Bulletin 6376 Rev A). Detailed protocol was previously described 17 (link). Primary antibodies were used as follow: rabbit anti-FOXG1 (Abcam, Cambridge, UK, ab18259, 1:1000); rabbit anti-caspase-9 (#9502, 1:1000), mouse anti-caspase-8 (#9746, 1:1000), rabbit anti-caspase-3 (#9665,1:1000), rabbit anti-cleaved caspase-3 (#9661, 1:1000), rabbit anti-AKT (#9272, 1:1000), mouse anti-ERK1/2 (#4696, 1:1000) and rabbit anti-c-Myc (#5605, 1:1000) were all from cell signaling (Danvers, MA, USA); Mouse anti-GAPDH was purchased from TransGen Biotech and mouse anti-α-tublin was get from Ray antibody Biotech (China).
5
Immunofluorescence Staining of Neural Markers
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At the time points
of interest,
cells were fixed in 4% paraformaldehyde in PBS for 20 min at room
temperature (RT) and washed with PBS. Cells were blocked for 45 min
at RT in blocking solution [0.1% Tween in PBS containing 2% bovine
serum albumin (BSA) and 20% goat serum]. Cells were washed three times
in PBS, and primary antibody was incubated for 1 h at RT. After washing
the cells three times with PBS, secondary antibody was applied with
Hoechst or DAPI (Invitrogen) and incubated for 1 h at RT in darkness.
Primary and secondary antibodies were mixed in staining solution (0.1%
Tween in PBS containing 5% goat serum). Cells were washed again with
PBS, fixed with ProLong Gold, and mounted on glass slides. The following
commercial antibodies were used: rabbit anti-tubulin-β3 (1:2000)
(Sigma), mouse anti-Isl-1 (1:1000) (DSHB), rabbit anti-FOXG1 (1:1000)
(Abcam), and mouse anti-TuJ1 (1:2000) (Covance). Alexa 488 and Alexa
546 conjugated secondary antibodies were obtained from Invitrogen.
of interest,
cells were fixed in 4% paraformaldehyde in PBS for 20 min at room
temperature (RT) and washed with PBS. Cells were blocked for 45 min
at RT in blocking solution [0.1% Tween in PBS containing 2% bovine
serum albumin (BSA) and 20% goat serum]. Cells were washed three times
in PBS, and primary antibody was incubated for 1 h at RT. After washing
the cells three times with PBS, secondary antibody was applied with
Hoechst or DAPI (Invitrogen) and incubated for 1 h at RT in darkness.
Primary and secondary antibodies were mixed in staining solution (0.1%
Tween in PBS containing 5% goat serum). Cells were washed again with
PBS, fixed with ProLong Gold, and mounted on glass slides. The following
commercial antibodies were used: rabbit anti-tubulin-β3 (1:2000)
(Sigma), mouse anti-Isl-1 (1:1000) (DSHB), rabbit anti-FOXG1 (1:1000)
(Abcam), and mouse anti-TuJ1 (1:2000) (Covance). Alexa 488 and Alexa
546 conjugated secondary antibodies were obtained from Invitrogen.
6
Immunohistochemical Characterization of Neuronal Differentiation
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Coverslips were fixed with 4% paraformaldehyde and immunohistochemistry was performed as previously described (Li et al., 2005 (link)). Antigen-antibody reactions were developed by appropriate fluorescence-conjugated secondary antibodies. Nuclei were visualized by Hoechst staining. Primary antibodies used in this study included mouse anti-Tra-1-60 (1:50; Santa Cruz Biotechnology), goat anti-Nanog (1:500; R&D), mouse anti-SSEA-4 (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti-HB9 (1:50; DSHB), rabbit anti-Foxg1 (1:100; Abcam), rabbit anti-Tbr1 (1:1000; Proteintech), mouse anti-βIII-tubulin (Tuj1; 1:100; DSHB) and rabbit anti-Tau (1:200; Sigma-Aldrich). The population of HB9-expressing neurons among total differentiated cells (Hoechst labeled) was counted as described previously (Li et al., 2009 (link)). Briefly, the Zeiss microscope was used to capture images. At least four fields of each coverslip were chosen and counted using ImageJ software (National Institutes of Health). For each group, six coverslips were counted. To quantify axonal swellings, blindly selected fields were imaged from six coverslips per group. The number of axonal swellings was counted (at least 500 neurites were analyzed per group) and divided by the total length of Tau+ axons in each field, which were measured using ImageJ software as we described before (Denton et al., 2014 (link)).
7
Immunocytochemistry of Neural Stem Cells
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NSCs, neurons and astrocytes cultured on coverslips were fixed with 4% paraformaldehyde for 15 min. After 3 washes in phosphate buffered saline, cells were permeabilized with 0.1% Triton for 20 min and blocked in 1% BSA for 30 min. Cells were incubated at 4°C overnight in primary antibody diluted in 1% BSA. The cells were then incubated in secondary antibody diluted in 1% BSA for 1 h at room temperature. The following primary antibodies were used: mouse anti-SOX2 (R&D Systems, Minneapolis, MN, USA), rabbit anti-nestin, mouse anti-PAX6, rabbit anti-FOXG1 (all from Abcam, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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