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Ab18259

Manufactured by Abcam
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Ab18259 is a monoclonal antibody that recognizes the BCL2L1 (BCL-X(L)) protein. BCL2L1 is a member of the BCL2 family of apoptosis regulator proteins. The antibody can be used for the detection of BCL2L1 in various applications.

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Lab products found in correlation

Ab31940, by Abcam (2 mentions) Alexa fluro 488 donkey anti chicken, by Jackson ImmunoResearch (2 mentions) Alexa fluro 647 donkey anti mouse, by Thermo Fisher Scientific (2 mentions) Ab22651, by Abcam (2 mentions) Ab13970, by Abcam (2 mentions) Ab8245, by Abcam (2 mentions) Ab18465, by Abcam (2 mentions) Ab12286, by Abcam (2 mentions) Ab126730, by Abcam (2 mentions) Mms 130r, by Fortrea (2 mentions) Ab21990, by Abcam (1 mentions) Ab195045, by Abcam (1 mentions) A2052, by Merck Group (1 mentions) Synapsin, by Abcam (1 mentions) Ab1511, by Merck Group (1 mentions) Nestin, by BD (1 mentions) Ab51841, by Abcam (1 mentions) Image studio, by LI COR (1 mentions) Odyssey clx, by LI COR (1 mentions) Gt anti sox1, by R&D Systems (1 mentions)

15 protocols using ab18259

1

Immunostaining of Neural Cell Cultures

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Cells (~ 1 × 105) were cultured on a cover glass in a 12‐well plate with 700 μL of medium. The neural cells were allowed to grow to desired morphology and density before staining procedure. Cells were first washed once with PBS and fixed by 4% paraformaldehyde/4% sucrose in PBS at room temp, followed by permeabilization and DNA denaturation by 0.2% TritonX‐100 in 4 m HCl. After that, the cells were washed with PBS and blocked in 80 μL BSA (3%). The cells were incubated with anti‐FOXG1 (ab18259; Abcam), Ki‐67 (BD, 550609), Otx1/2 (ab21990; Abcam, Cambridge, MA, USA), PAX6 (ab195045; Abcam), NESTIN (BD, 561230), Nkx2.1 (MAB5460; Millipore, Darmstadt, Germany), MAP2 (M4403; Sigma, St. Louis, MO, USA), GABA (A2052; Sigma) VGAT (131011; Synaptic systems, Goettingen, Germany), SYNAPSIN (Abcam), TBR1 (ab31940; Abcam), GAT1 and Glutamate (ab1511; Millipore) in BSA (3%) at 4 °C overnight, and then conjugated with and Hoechst 33342 or DAPI. The glass slides were mounted with a cover slip before imaging.
Tu J., Cao D., Li L., Cheung H, & Chan W. (2018). MicroRNA profiling during directed differentiation of cortical interneurons from human‐induced pluripotent stem cells. FEBS Open Bio, 8(4), 502-512.
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2

Foxg1 Protein Quantification in Embryonic Brain

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Protein lysates for western blotting were prepared by homogenizing whole brains isolated from e16.5 FoxG1MUT/WT embryos in Tris lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 μM Pepstatin, 10 μM Leupeptin, 200 μM PMSF). Protein concentration was determined using the 660nm protein assay (Pierce 22660). 5 ug of total protein for each sample was separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane using standard methods. Following western transfer, membranes were incubated with primary antibodies (1:1000 rabbit anti-Foxg1 (Abcam ab18259) and 1:5000 mouse anti-TBP (Abcam ab51841)) overnight at 4 degrees C. The following day, membranes were incubated with secondary antibodies (1:10,000 goat anti-rabbit 800CW (Licor 926–32211) and 1:10,000 goat anti-mouse 680RD (Licor 926–68070)) for 1 hour prior to imaging on a Licor Odyssey CLX. Quantification was performed using Licor Image Studio.
Erickson K.R., Farmer R., Merritt J.K., Miletic Lanaghan Z., Does M.D., Ramadass K., Landman B.A., Cutting L.E, & Neul J.L. (2022). Behavioral and brain anatomical analysis of Foxg1 heterozygous mice. PLoS ONE, 17(10), e0266861.
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3

Quantifying Neural Progenitor Markers

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At D11 or D21, cells were plated onto Greiner Bio-One Cellstar 96-well μclear Flat Bottomed Microplates (Fisher Scientific 07-000-166) for immunocytochemistry at a density of 90,000 cells/cm2. Cells were fixed 24 hours later with 4% paraformaldehyde for 30 minutes at room temperature. Cells were permeabilized in PBS with 0.2% Triton X-100 (PBS+) for 20 minutes and blocked in PBS with 5% donkey serum and 0.05% TritonX-100 (PBS++) for 2 hours. Primary antibodies were diluted in PBS++ and incubated on cells overnight at 4°C. Primary antibodies included: gt anti-SOX1 (R&D Systems AF3369) diluted 1:100, rb anti-PAX6 (BioLegend 901301) diluted 1:200, rb anti-Ki67 (Abcam Ab16667) diluted 1:100, rabbit anti-FOXG1 (Abcam AB18259) diluted 1:100, and mouse anti-MAP2 (ThermoFisher Scientific 131500) diluted 1:500. Secondaries used included: Alexa Fluor 488 AffiniPure donkey anti-goat IgG, donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories 715-545-150), and donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories 715-545-152). Secondaries were diluted 1:800 in PBS++ and incubated for 3 hours at room temperature. Cells were incubated for 5 minutes with Hoechst nuclear dye (ThermoFisher Scientific H3570) at 2μg/mL and stored in PBS, protected from light, at 4°C until imaging.
Prince L.M., Neely M.D., Warren E.B., Thomas M.G., Henley M.R., Smith K.K., Aschner M, & Bowman A.B. (2021). Environmentally Relevant Developmental Methylmercury Exposures Alter Neuronal Differentiation in a Human-Induced Pluripotent Stem Cell Model. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 152, 112178.
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4

Comprehensive Immunofluorescence Labeling Protocol

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Immunofluorescence was performed as previously described [19 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500); anti-Calbindin (Millipore, AB1778, 1:250); anti-Foxg1 (Abcam, ab18259, 1:1000); anti-GFP (Abcam, ab13970, 1:1000); anti-L1 (Millipore, MAB5272, 1:500); anti-Pax6 (BioLegend, 901,301, 1:1000); and anti-Vglut2 (Millipore, MAB5504, 1:500). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703–545-155, 1:500), Alexa Fluor 488 donkey anti-rabbit (Life, A21206, 1:500), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500), Alexa Fluor 488 donkey anti-rat (Life, A21208, 1:500), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500) and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500).
Liu B., Zhou K., Wu X, & Zhao C. (2018). Foxg1 deletion impairs the development of the epithalamus. Molecular Brain, 11, 5.
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5

Immunocytochemistry of Cultured Neurons

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Cultured induced neurons were fixed in 4% paraformaldehyde + 20% sucrose in DPBS for 20 min at room temperature. Cells were incubated with blocking buffer containing 4% horse serum, 0.1 M Glycine, and 0.3% Triton-X in PBS for 1 hour at room temperature. Primary antibodies, diluted in 4% horse serum in PBS, were incubated overnight at 4oC. Secondary antibodies were diluted in 4% horse serum and applied for 1 hour at room temperature. Samples were washed 3x with PBS and imaged on spinning disc confocal microscope (Andor Dragonfly) with a 20x air objective. The following antibodies were used: rabbit anti-SV2A (1:1000, Abcam ab32942), chicken anti-MAP2 (1:10,000, Abcam ab5392), rabbit anti-Synaptotagmin-11 (1;1000, Synaptic Systems 270 003), mouse anti-hNA (1:1000, Millipore MAB1281), rabbit anti-Cux1 (1:500, Santa Cruz m-222), guinea pig anti-NeuN (1:1000, Synaptic Systems 266004), mouse anti-Sox2 (1:500, R&D systems, MAB2018), mouse anti-Sox1 (1:500, R&D systems, AF3369), mouse anti-Ki67 (1:500, BD Biosciences 550609), rabbit anti-FoxG1 (1:500, abcam ab18259). Alexafluor plus-555 and Alexafluor plus-488 conjugated secondary antibodies (1:5,000) were obtained from Invitrogen.
Nehme R., Pietiläinen O., Artomov M., Tegtmeyer M., Valakh V., Lehtonen L., Bell C., Singh T., Trehan A., Sherwood J., Manning D., Peirent E., Malik R., Guss E.J., Hawes D., Beccard A., Bara A.M., Hazelbaker D.Z., Zuccaro E., Genovese G., Loboda A.A., Neumann A., Lilliehook C., Kuismin O., Hamalainen E., Kurki M., Hultman C.M., Kähler A.K., Paulo J.A., Ganna A., Madison J., Cohen B., McPhie D., Adolfsson R., Perlis R., Dolmetsch R., Farhi S., McCarroll S., Hyman S., Neale B., Barrett L.E., Harper W., Palotie A., Daly M, & Eggan K. (2022). The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia. Nature Communications, 13, 3690.
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6

Immunohistochemical analysis of neural progenitor cells

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Upon completion of timelapse imaging, brain slices were fixed in 4% PFA in PBS overnight at 4 °C and then transferred to PBS. Slices were subjected to boiling citrate-based antigen retrieval solution for 20 min and permeabilized and blocked in blocking buffer (PBS plus 0.1% Triton X-100, 10% serum, and 0.2% gelatin) for 1 h. Primary antibodies were diluted in blocking buffer and applied to slices for 36 h at 4 °C. Slices were washed with PBS plus 0.5% Triton X-100 and then incubated in secondary antibodies diluted in blocking buffer for 3–5 h. Images were acquired on a Leica TCS SP5 X laser confocal microscope. Primary and secondary antibodies used: goat anti-SOX2 (Santa Cruz Biotechnology, sc-17320, 1:250), rabbit anti-TBR2 (Abcam, ab23345, 1:100), chicken anti-GFP (Aves Labs, GFP-1020, 1:1,000), rabbit anti-FOXG1 (Abcam, ab18259, 1:250), mouse anti-pVIM (Abcam, ab22651, 1:250), mouse anti-phosphohistoneH3 (Abcam, ab14955, 1:200), AlexaFluor 488, 546, 594, or 647-conjugated donkey anti-goat, -rabbit, -mouse IgG (Invitrogen, 1:500), and AlexaFluor 488 donkey anti-chicken IgY (Jackson ImmunoResearch, 703-545-155, 1:500).
Subramanian L., Bershteyn M., Paredes M.F, & Kriegstein A.R. (2017). Dynamic behaviour of human neuroepithelial cells in the developing forebrain. Nature Communications, 8, 14167.
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7

Immunohistochemical Analysis of Hippocampal Neurons

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Brains were perfused and post-fixed with 4% paraformaldehyde at 4 °C for 12–16 h, cryoprotected in 30% sucrose and embedded in optimum cutting temperature compound (OCT). Coronal sections (20 μm thick) were obtained using a Leica cryostat (CM 3050S). Immunostaining was performed as previously described [11 (link)]. Chicken anti-GFP (Abcam, ab13970, 1:500) and rabbit anti-FOXG1 (Abcam, AB18259, 1:500) were used as primary antibodies, and Alexa Fluor 488 goat anti-chicken IgG (Molecular Probes, A11039, 1:500) and Alexa Fluor 546 donkey anti-rabbit IgG(Molecular Probes, A10040, 1:500) were used as secondary antibodies. DAPI (Sigma, D9564, 1:1000) was incubated for 15 min (mins) before coverslips were applied. Images were captured by a confocal microscope (Olympus, FV1000). For cell counting, three brains from each genotype from at least two different litters were collected and images of two consecutive hippocampal slices of each brain were used; the dorsal hippocampus was outlined using ImageJ software (NIH), and CA1 cell numbers were counted manually based on the DAPI staining.
Yu B., Liu J., Su M., Wang C., Chen H, & Zhao C. (2019). Disruption of Foxg1 impairs neural plasticity leading to social and cognitive behavioral defects. Molecular Brain, 12, 63.
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8

Protein Quantification and Western Blot Analysis

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OC-1 cells and cochleae were lysed with cold RIPA Lysis Buffer plus PMSF. Nuclear and cytosolic protein fractions were extracted using a Nuclear/Cytosol Fractionation Kit (BioVision, K266) according to the manufacturer's protocol. A BCA Protein Quantification Kit (Beyotime Biotechnology, P0010) was used to measure the protein concentration with GAPDH as the reference protein in line with the manufacturer's instructions. LC3B-II was measured using an anti-LC3B rabbit polyclonal antibody (Sigma-Aldrich, L7543), FoxG1 was measured using an anti-FoxG1 rabbit polyclonal antibody (Abcam, ab18259), and GAPDH was measured using an anti-GAPDH rabbit polyclonal antibody (Abcam, ab8245). Peroxidase-conjugated goat anti-rabbit immunoglobulin G (Abcam, ab6721) was used as the secondary antibody. The proteins were bound to polyvinylidene fluoride membranes, and a SuperSignal West Dura chemiluminescent substrate kit was used to detect the complexes according to the manufacturer's instructions. The western blots were semi-quantified using Image J to measure the intensities of the bands.
He Z.H., Zou S.Y., Li M., Liao F.L., Wu X., Sun H.Y., Zhao X.Y., Hu Y.J., Li D., Xu X.X., Chen S., Sun Y., Chai R.J, & Kong W.J. (2019). The nuclear transcription factor FoxG1 affects the sensitivity of mimetic aging hair cells to inflammation by regulating autophagy pathways. Redox Biology, 28, 101364.
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9

Chromatin Immunoprecipitation Protocol

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Chromatin was prepared and immunoprecipitation was undertaken according to protocols described previously (Sofueva et al. 2013 (link)). Sonication was performed in 0.7% SDS using a Diagenode Bioruptor (maximum power 30 sec on and 30 sec off for 45 min). Pull-down was undertaken using Dynabead protein G sepharose beads (Thermo Scientific) conjugated with 10 µL of ChIP-grade antibody (anti-FoxG1 [Abcam, ab18259] and anti-V5 [Abcam, ab15828]) diluted in 250 µL of buffer.
Bulstrode H., Johnstone E., Marques-Torrejon M.A., Ferguson K.M., Bressan R.B., Blin C., Grant V., Gogolok S., Gangoso E., Gagrica S., Ender C., Fotaki V., Sproul D., Bertone P, & Pollard S.M. (2017). Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators. Genes & Development, 31(8), 757-773.
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10

Immunofluorescence Staining Protocol

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Immunofluorescence was performed as previously described [21 (link),22 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500, Billerica, MA, USA); anti-Ctip2 (Abcam, ab18465, 1:2000, Cambridge, MA, USA); anti-Foxg1 (Abcam, ab18259, 1:1000, Cambridge, MA, USA); anti-GFP (Abcam, ab13970, 1:1000, Cambridge, MA, USA); anti-Lhx2 (Abcam, ab184337, 1:500, Cambridge, MA, USA); anti-P73 (Abcam, ab40658, 1:500, Cambridge, MA, USA); anti-Prox1 (Millipore, AB5475, 1:1000, Billerica, MA, USA); and anti-Reelin (Millipore, MAB5364, 1:1000, Billerica, MA, USA). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703-545-155, 1:500, West Grove, PA, USA), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500, Gaithersburg, MD, USA), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500, Gaithersburg, MD, USA), Alexa Fluor 546 donkey anti-rat (Life, A10040, 1:500, Gaithersburg, MD, USA), CF 568 donkey anti-rat (Sigma-Aldrich, SAB4600077, 1:500, St. Louis, MO, USA), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500, St. Louis, MO, USA), and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500, Carlsbad, CA, USA).
Liu B., Xiao H, & Zhao C. (2018). Forced Expression of Foxg1 in the Cortical Hem Leads to the Transformation of Cajal-Retzius Cells into Dentate Granule Neurons. Journal of Developmental Biology, 6(3), 16.
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