mouse  (Developmental Studies Hybridoma Bank)

Journal: Epilepsia

Article Title: kcnb1 loss of function in zebrafish causes neurodevelopmental and epileptic disorders associated with γ‐aminobutyric acid dysregulation

doi: 10.1111/epi.18407

Figure Lengend Snippet: kcnb1 is expressed in distinct cell subtypes and various regions of the central nervous system in 6 days post fertilization (dpf) wild‐type (WT) zebrafish. (A, B) Horizontal and transversal sections of WT zebrafish expressing cells (4,6‐diamidino‐2‐phenylindole [DAPI]; blue) labeled with anti‐kcnb1 (green), showing a large expression of the protein in the central nervous system (CNS) at 6 dpf. The protein is expressed in the telencephalon, diencephalon, midbrain including the optic tectum, and hindbrain comprising the cerebellum and the spinal cord (A: scale bar = 50 μm, magnification = 10×; B: scale bar = 30 μm, magnification 20×). (C) Horizontal section of a WT zebrafish at 6 dpf showing the presence of kcnb1 along the spinal cord (scale bar = 10 μm, magnification = 63×). (D–F) Horizontal sections of 6‐dpf WT zebrafish expressing cells (DAPI; blue), kcnb1 (green), and specific cell subtype markers, respectively. (D) A neuronal nuclear marker (neuronal nuclear antigen [NeuN]; red, scale bar = 30 μm, magnification = 20×), (E) oligodendrocyte transcription factor 2 (Olig2; red, scale bar = 10 μm, magnification = 63×), and (F) CX3C motif chemokine receptor 1 expressed in microglial cells (CX3CR1; red, scale bar = 10 μm, magnification = 63×). Images demonstrate the colocalization between kcnb1 and the three different cell subtype markers, represented in white and marked by arrows. These results indicate the presence of kcnb1 in neurons, oligodendrocytes, and microglial cells. The colocalization was determined using Z ‐stack projection with IMARIS v10.1.0 software (Oxford Instruments). In figures, CNS regions are delimited by dotted lines. D, diencephalon; E, eyes; H, hindbrain; M: midbrain; T, telencephalon. n = 3–4/section.

Article Snippet: After permeabilization for 30 min, the slices were incubated overnight at 4°C in blocking solution containing primary antibodies: Kcnb1 (1:100, Tebubio, #PAB7569), NeuN (1:100, Merck, #ABN90), Olig2 (1:100, DSHB, #PCRP‐OLIG2‐1E9‐s), CX3CR1 (1:100, Proteintech, #13885‐1‐AP), Ankyrin G (1:100, Proteintech, #27980‐1‐AP), and HuC (1:100, Tebubio, #FNab04072).

Techniques: Expressing, Labeling, Marker, Software

Journal: Experimental neurology

Article Title: Ethanol deregulates Mecp2 /MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

doi: 10.1016/j.expneurol.2015.01.006

Figure Lengend Snippet: The list of primers used for qRT-PCR experiments and their sources.

Article Snippet: For cell population determination, approximately 250 DAPI + cells from D2 and 400–500 DAPI + cells from D8 collectively from 3 biological replicates were counted using ImageJ program, as described previously ( Liyanage et al., 2013 ). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Primary antibody Application and dilution Molecular weight (kDa) Description Source MeCP2 (C-terminal) WB 1:1000 72 Mouse monoclonal Millipore, 07-013 Beta (β) ACTIN WB: 1:2500 42 Mouse monoclonal Sigma, A2228 GAPDH WB: 1:500 37 Rabbit polyclonal Santa Cruz, Sc-25778 GFAP IF 1:200 N/A Mouse monoclonal Invitrogen, 421262 Beta (β) TUBULIN III (TUB III) IF 1:200 N/A Mouse monoclonal Chemicon, MAB1637 MBP IF 1:100 N/A Rabbit polyclonal Abcam, ab40390 5mC Dot blot 1:1000 N/A Mouse monoclonal Abcam, Ab73938 5hmC Dot blot 1:1000 N/A Rabbit polyclonal Active Motif, 39769 NESTIN IF 1:200 N/A Rat polyclonal Developmental Studies Hybridoma Bank, Rat-401c KI67 IF 1:200 N/A Rabbit polyclonal Sc-15402 OLIG2 IF 1:200 N/A Rabbit polyclonal Millipore, AB9610 RNA Polymerase (Pol) II WB 1:1000 217 Mouse monoclonal Abcam, Ab817 HDAC2 WB 1:1000 55 Mouse monoclonal Abcam, Ab12169 Alpha (α) TUBULIN WB 1:1000 50 Mouse monoclonal Sigma, T9026 Open in a separate window List of primary antibodies used in western blot and immunocytochemistry. table ft1 table-wrap mode="anchored" t5 Table 3 caption a7 Secondary antibody Application and dilution Source Alexa Fluor 488 conjugated goat anti-mouse IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11017","term_id":"489238","term_text":"A11017"}} A11017 Alexa Fluor 594 conjugated goat anti rabbit IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 Peroxidase-AffiniPure Gt anti-mouse IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 115-035-174 Peroxidase-AffiniPure donkey anti-rabbit IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 711-035-152 Open in a separate window List of secondary antibodies used in western blot and immunocytochemistry.

Techniques: Sequencing

Journal: Experimental neurology

Article Title: Ethanol deregulates Mecp2 /MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

doi: 10.1016/j.expneurol.2015.01.006

Figure Lengend Snippet: List of primary antibodies used in western blot and immunocytochemistry.

Article Snippet: For cell population determination, approximately 250 DAPI + cells from D2 and 400–500 DAPI + cells from D8 collectively from 3 biological replicates were counted using ImageJ program, as described previously ( Liyanage et al., 2013 ). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Primary antibody Application and dilution Molecular weight (kDa) Description Source MeCP2 (C-terminal) WB 1:1000 72 Mouse monoclonal Millipore, 07-013 Beta (β) ACTIN WB: 1:2500 42 Mouse monoclonal Sigma, A2228 GAPDH WB: 1:500 37 Rabbit polyclonal Santa Cruz, Sc-25778 GFAP IF 1:200 N/A Mouse monoclonal Invitrogen, 421262 Beta (β) TUBULIN III (TUB III) IF 1:200 N/A Mouse monoclonal Chemicon, MAB1637 MBP IF 1:100 N/A Rabbit polyclonal Abcam, ab40390 5mC Dot blot 1:1000 N/A Mouse monoclonal Abcam, Ab73938 5hmC Dot blot 1:1000 N/A Rabbit polyclonal Active Motif, 39769 NESTIN IF 1:200 N/A Rat polyclonal Developmental Studies Hybridoma Bank, Rat-401c KI67 IF 1:200 N/A Rabbit polyclonal Sc-15402 OLIG2 IF 1:200 N/A Rabbit polyclonal Millipore, AB9610 RNA Polymerase (Pol) II WB 1:1000 217 Mouse monoclonal Abcam, Ab817 HDAC2 WB 1:1000 55 Mouse monoclonal Abcam, Ab12169 Alpha (α) TUBULIN WB 1:1000 50 Mouse monoclonal Sigma, T9026 Open in a separate window List of primary antibodies used in western blot and immunocytochemistry. table ft1 table-wrap mode="anchored" t5 Table 3 caption a7 Secondary antibody Application and dilution Source Alexa Fluor 488 conjugated goat anti-mouse IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11017","term_id":"489238","term_text":"A11017"}} A11017 Alexa Fluor 594 conjugated goat anti rabbit IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 Peroxidase-AffiniPure Gt anti-mouse IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 115-035-174 Peroxidase-AffiniPure donkey anti-rabbit IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 711-035-152 Open in a separate window List of secondary antibodies used in western blot and immunocytochemistry.

Techniques: Western Blot, Immunocytochemistry, Molecular Weight, Dot Blot

Journal: Experimental neurology

Article Title: Ethanol deregulates Mecp2 /MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

doi: 10.1016/j.expneurol.2015.01.006

Figure Lengend Snippet: Differentiating neural stem cell (NSC) model. A) Schematic representation of NSC isolation, culture and differentiation. NSC are isolated from embryonic day (E) 14.5 forebrain (a) and cultured in the presence of growth factors (EGF and FGF) to generate neurospheres. (b) The neurospheres are dissociated at day zero (D0) (c) and differentiated for 8 days. Samples were collected at early stage of differentiation at day (D) 2 (d) and at a later differentiation stage D8 (e). Scale bars represent 20 μm. B) Representative images of different cell types in D2 and D8 population. a–a′: Ki67, b–b′: NESTIN, c–c′: OLIG2, d–d′: β-TUB III, e–e′: GFAP, f–f′: MBP. Note that no MBP+ cells were found at D2 (f). Scale bars represent 20 μm. C) Quantified percentages of the cellular composition of D2 and D8 differentiating populations. N = 3 ± SEM. D) MeCP2 expression during NSC differentiation detected by western blot. CE: Cytoplasmic extracts and NE: nuclear extracts. GAPDH was used as the loading control. N = 3 ± SEM.

Article Snippet: For cell population determination, approximately 250 DAPI + cells from D2 and 400–500 DAPI + cells from D8 collectively from 3 biological replicates were counted using ImageJ program, as described previously ( Liyanage et al., 2013 ). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Primary antibody Application and dilution Molecular weight (kDa) Description Source MeCP2 (C-terminal) WB 1:1000 72 Mouse monoclonal Millipore, 07-013 Beta (β) ACTIN WB: 1:2500 42 Mouse monoclonal Sigma, A2228 GAPDH WB: 1:500 37 Rabbit polyclonal Santa Cruz, Sc-25778 GFAP IF 1:200 N/A Mouse monoclonal Invitrogen, 421262 Beta (β) TUBULIN III (TUB III) IF 1:200 N/A Mouse monoclonal Chemicon, MAB1637 MBP IF 1:100 N/A Rabbit polyclonal Abcam, ab40390 5mC Dot blot 1:1000 N/A Mouse monoclonal Abcam, Ab73938 5hmC Dot blot 1:1000 N/A Rabbit polyclonal Active Motif, 39769 NESTIN IF 1:200 N/A Rat polyclonal Developmental Studies Hybridoma Bank, Rat-401c KI67 IF 1:200 N/A Rabbit polyclonal Sc-15402 OLIG2 IF 1:200 N/A Rabbit polyclonal Millipore, AB9610 RNA Polymerase (Pol) II WB 1:1000 217 Mouse monoclonal Abcam, Ab817 HDAC2 WB 1:1000 55 Mouse monoclonal Abcam, Ab12169 Alpha (α) TUBULIN WB 1:1000 50 Mouse monoclonal Sigma, T9026 Open in a separate window List of primary antibodies used in western blot and immunocytochemistry. table ft1 table-wrap mode="anchored" t5 Table 3 caption a7 Secondary antibody Application and dilution Source Alexa Fluor 488 conjugated goat anti-mouse IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11017","term_id":"489238","term_text":"A11017"}} A11017 Alexa Fluor 594 conjugated goat anti rabbit IgG IF 1:2000 or 1:3000 Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A11037","term_id":"492397","term_text":"A11037"}} A11037 Peroxidase-AffiniPure Gt anti-mouse IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 115-035-174 Peroxidase-AffiniPure donkey anti-rabbit IgG WB 1:7500, Dot blot 1:7500 Jackson ImmunoResearch 711-035-152 Open in a separate window List of secondary antibodies used in western blot and immunocytochemistry.

Techniques: Isolation, Cell Culture, Expressing, Western Blot