The cultured cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) and used for immunostaining. Fixed cells were blocked with 5% normal goat serum in phosphate-buffered saline and 0.1% Triton X-100 (PBST) and then incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: rat anti-GFP antibody (1/500, Nacalai Tesque, 04404-84), monoclonal anti-α-tubulin antibody (1/500, Sigma, T9026), rat anti-myelin basic protein (MBP) antibody (Millipore, MAB386), monoclonal O4 antibody (1/300, R&D systems, MAB1326) and rabbit anti-paxillin antibody (1/250, abcam, ab32084, clone Y113). After being rinsed with PBST, the cells were incubated with secondary antibodies. The secondary antibodies used were Alexa 488- or 594-conjugated goat anti-mouse, anti-rabbit and anti-rat IgG or goat anti-mouse IgM (Molecular Probes). Fluorescent signals were visualized using AX70 fluorescence microscope (Olympus, Tokyo) and A1Rsi confocal fluorescence microscope (Nikon, Tokyo). The number of OL primary processes was analyzed using “Analyze/Sholl” tool of Fiji software based on ImageJ (NIH).
Rat anti gfp antibody
Manufactured by Nacalai Tesque
Sourced in Japan
The Rat anti-GFP antibody is a laboratory reagent used to detect and identify the presence of green fluorescent protein (GFP) in experimental samples. It is a specific antibody raised in rats against the GFP molecule, which can be used to label and visualize GFP-expressing cells or proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Monoclonal anti α tubulin antibody, by Merck Group (1 mentions)
Ax70 fluorescence microscope, by Olympus (1 mentions)
Goat anti mouse igm, by Thermo Fisher Scientific (1 mentions)
Mab1326, by Abcam (1 mentions)
A1rsi confocal fluorescence microscope, by Nikon (1 mentions)
Salmon sperm dna, by Merck Group (1 mentions)
Alexa 488 conjugated antirat igg, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti dsred antibody, by Takara Bio (1 mentions)
Fv1200, by Olympus (1 mentions)
Mouse anti neun antibody, by Merck Group (1 mentions)
Anti mouse cy5 antibody, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
7 protocols using rat anti gfp antibody
1
Immunostaining and Imaging of Oligodendrocytes
2
Protein-DNA Interaction Analysis via EMSA
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Electrophoretic mobility shift assays (EMSAs) were performed using 32P-labeled double-stranded oligonucleotides (Supplementary Figure S3D ) as described (15 (link)). An amount of 0.05 μg salmon sperm DNA (Sigma, D1626), sheared to 100–400 bp fragments, was used as an unspecific competitor and the probes were either incubated without cell lysate, with control HEK293T lysates (obtained from cells transfected with EGFP vector or empty pCMV5) or myc-Cterm- or EGFP-Cterm-containing whole cell lysates. For super-shift,1 μl of a rat anti-GFP antibody (Nacalai Tesque, 04404–84, Lot No. M7E5845) was preincubated with cell extracts for 20 min prior to addition of the labelled probes.
3
Quantifying Interneurons in Somatosensory Cortex
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Serial 40-µm-thick sections were cut in the coronal direction from the somatosensory cortex of GAD67-green fluorescent protein (GFP) knock-in mice (Tamamaki et al., 2003 (link); n = 5 mice) and were triple-immunostained for PV, VGluT2, and GFP. Two sections at different rostrocaudal positions were selected from each animal, one at the bregma and the other located 1.7 mm caudal to the bregma, to detect anterolateral and posteromedial barrels, respectively. Detection of GFP-expressing cells was performed using rat anti-GFP antibody (Nacalai Tesque; catalog#04404-84, RRID:AB_10013361, dilution 1:1,000) followed by labeling with Alexa 488–conjugated antirat IgG (Jackson ImmunoResearch; catalog #712-545-153, RRID:AB_2340684; dilution 1:250); labeling for PV and VGluT2 was performed simultaneously with GFP labeling by the same method as in triple labeling for PV, VGLuT2, and NeuN described above. Three barrels and intervening septal areas were reconstructed in each rostral and caudal section in each mouse by tracing the contour in every other optical slice using CLSM images acquired with a 20× objective, followed by cell counting and calculation of PV/GAD67 proportion as in the measurement of PV/NeuN proportion.
4
Immunofluorescence Staining of Cryo-Embedded Tissue
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For immunofluorescence staining, tissues fixed with 4% paraformaldehyde in PBS were cryo-embedded in OCT compound (Sakura Finetechnical) and cut into 7-μm-thick sections. Nuclei were counterstained with Hoechst 33342 dye. Specimens were observed with a microscope (Olympus IX 73; Olympus, Tokyo, Japan). The following were used as primary antibodies: rat anti-GFP antibody (1:1,000, Nakalai Tesque, Inc., Kyoto, Japan), mouse anti-SCP3 antibody (1:200, Abcam) and rabbit anti-peanut agglutinin antibody (1:400, Life technologies). The secondary antibodies used were mouse anti-mouse IgG, goat anti-rabbit IgG, and goat anti-rat IgG, conjugated with Alexa 488 or Alexa 555 (1:200; Life technologies).
5
Evaluating CNTNAP2 and AHI1 Knockdown
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We used the HEK293T cell system70 (link),75 (link) to evaluate the efficacy of CNTNAP2 or AHI1 knockdown. HEK293T cells in a 24-well dish were transfected with an mOrange2-fused cDNA (CNTNAP2-mOrange or AHI1-mOrange) together with one of the following vector, an RNAi-knockdown (CNTNAP2-knockdown or AHI1-knockdown) vector, a rescue (CNTNAP2-rescue or AHI1-rescue) vector, or a scramble (CNTNAP2-Scr or AHI1-Scr) vector, using X-tremeGENE 9 reagents. One day later, these cells were fixed. After permeabilization by Triton-X-100 and blocking, primary antibodies (a Rat anti-GFP antibody, Nacalai Tesque; a Rabbit anti-DsRed antibody, Clontech laboratories inc.) were applied. Fluorescence signals from these cells were examined under a confocal laser scanning microscope (FV1200, Olympus).
6
Immunofluorescent Neuronal Characterization
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Mice were deeply anesthetized with pentobarbital (100 μg/g of body weight, intraperitoneal injection), perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer and then processed to obtain coronal sections (100 μm in thickness). After permeabilization (0.5% Triton-X) and blockade of nonspecific binding (10% donkey serum), a rat anti-GFP antibody (1:1000; Nacalai Tesque, Kyoto, Japan), a mouse anti-CaMKII antibody (1:200; Abcam, Cambridge, UK) and a mouse anti-NeuN antibody (1:1000; MerckMillipore, Darmstadt, Germany) were applied overnight at 4 °C. After incubation with secondary antibodies for 4 h at room temperature (an anti-rat IgG Alexa Fluor 488 antibody; 1:400, Life Technologies, Inc. or an anti-mouse Cy5 antibody; 1:300, Jackson), the immunolabeled sections were counterstained with DAPI (300 nM; Invitrogen, Life Technologies, Inc.) for nucleic acid staining.
7
Dual Immunofluorescence Staining of Microtubules and GFP
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Cells were fixed as described in Kushida et al. (2011) . For double detection of MTs and GFPs, fixed cells were treated with the mouse anti-chicken a-tubulin antibody (Cat# CP06, Calbiochem, Darmstadt, Germany; 1/300 dilution) and the rat anti-GFP antibody (Cat# 04404-26, Nacalai tesque, Kyoto, Japan; 1/300 dilution) at room temperature, overnight. After washing, cells were incubated with the rhodamine-conjugated goat anti-mouse IgG antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA; 1/300 dilution) and the Alexa Fluor 488-conjugated donkey anti-rat IgG antiserum (Life technologies, Junction City, OR; 1/300 dilution) for 6 hr. DNA was stained with DAPI (0.5 lg/ml). Cells were observed under a confocal laser scanning microscope, LSM700 (Carl Zeiss, Oberkochen, Germany) with an alpha Plan-Apochromat 1009 lens (N. A. 1.46) . For single staining of MTs, fixed cells were treated with the mouse anti-chicken a-tubulin antibody (described above, 1/150 dilution) followed by the incubation with FITC-conjugated goat anti-mouse IgG antiserum (TAGO, Burlingame, CA; 1/150 dilution). DNA was stained with 24 lg/ml propidium iodide for 40 min after the treatment with 0.1 mg/ml RNase for 1 h. Cells were observed under a confocal microscope, LSM510 (Carl Zeiss, Oberkochen, Germany) with an alpha Plan-Fluar 1009 lens (N.A. 1.45).
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