Rabbit anti gfp

Manufactured by Abcam

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Rabbit anti-GFP is a primary antibody that binds to green fluorescent protein (GFP). It is used for the detection and localization of GFP in various experimental applications.

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173 protocols using rabbit anti gfp

Antibody Protocol for Western Blotting and Immunofluorescence

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The following primary antibodies were used for Western blotting and diluted in TBS-Tween containing 1% dry-milk or 3% BSA: mouse anti-gephyrin (clone 3B11 cell culture supernatant, 1∶50); rabbit anti–PSD-95 (1∶500, Synaptic Systems); rabbit anti–GABA_A-α1, guinea-pig anti–GABA_A-α5, guinea-pig anti–GABA_A-γ2 subunit (all 1∶500, Jean-Marc Fritschy, University of Zurich); rabbit anti–GABA_A-γ2 (1∶500, Synaptic Systems); mouse anti–GABA_A-β2/3 (1∶200, Millipore); rabbit anti–β-tubulin (1∶150, Santa Cruz); rabbit anti-GFP (1∶3,000, Abcam); mouse anti–HA-tag (1∶1,000, clone 12CA5, Roche); and mouse anti–myc-tag (1∶10 cell culture supernatant, clone 9E10). HRP secondary antibodies (Santa Cruz) were used in 1∶5,000 dilutions in TBS-Tween containing 1% dry-milk. Streptavidin HRP conjugates were from Cell Signaling.
The following antibodies were used for immunofluorescence and PLA assay: mouse anti-gephyrin (1∶50 cell culture supernatant, clone 3B11 [53] (link)); rabbit anti-VGAT (1∶500, Synaptic Systems); mouse anti–PSD-95 (1∶500, Thermo Scientific); rabbit anti–GABA_A-γ2 and α2 (1∶500, Synaptic Systems); mouse anti–HA-tag (1∶1,000, clone 12CA5, Roche); mouse anti–myc-tag (1∶10 cell culture supernatant, clone 9E10); rabbit anti-giantin (1∶1,000, Abcam); and rabbit anti-GFP (1∶1,000, Abcam). Secondary antibodies were all goat-raised Alexa Fluor 488 or 568 antibodies (Life Technologies).

Dejanovic B., Semtner M., Ebert S., Lamkemeyer T., Neuser F., Lüscher B., Meier J.C, & Schwarz G. (2014). Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses. PLoS Biology, 12(7), e1001908.

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Fluorescent Staining of Nervous Tissues

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Adult brains were dissected and fixed in 4% paraformaldehyde at room temperature for 20 minutes. Late third-instar larval NMJs were dissected in Schneider's insect medium (Sigma-Aldrich) and fixed with Bouin's solution (Sigma-Aldrich) for 5 min. All samples were then stained by standard methods: washed 3× in PBS containing 0.3% Triton-X 100 (PBT), blocked for 30 min in 5% normal goat serum, incubated with primary antibodies at 4°C overnight, washed 3× with PBT, incubated with secondary antibodies at 4°C overnight, washed 3× with PBT, and mounted in Vectashield (Vector Laboratories). We used the following primary antibodies from the Developmental Studies Hybridoma Bank (DSHB): anti-Chp (24B10, at dilution 1:200); anti-Dlg (4F3, 1:250); anti-CSP (6D6, 1:250); anti-Syt (3H2 2D7, 1:250); and anti-Brp (nc82, 1:100). Other primary antibodies used were: rabbit anti-GFP (1:1000; Abcam); fluorescence-conjugated anti-HRP (1:250; Jackson Immuno Labs); rabbit anti-GFP (1:1000; Abcam); rabbit anti-pMAD (1:500, a gift from Carl Heldin; Persson et al. 1998) ; and rabbit anti-GluRIIC (1:2500, a gift from Aaron DiAntonio; Marrus et al. 2004) . All secondary antibodies were goat IgG coupled to Alexa Fluor 488, Alexa Fluor 555, or AlexFluor 633 (1:500; Thermo Fisher).

Spinner M.A., Pinter K., Drerup C.M., & Herman T. (2020). Vezatin is required for the retrograde axonal transport of endosomes in Drosophila and zebrafish.

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Immunohistochemistry of SFO and OVLT

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The desired brain sections containing the SFO and OVLT were initially washed free‐floating in PBS. Next, brain tissue was permeabilized with 0.1% Triton X100 (Sigma) for 20 minutes at room temperature (RT), following further washing in PBS. Slices were then blocked in 5% normal donkey serum (NDS; Sigma) and 0.05% Triton X100 at RT for 30 minutes. A 48 hours incubation at 4°C in a PBS solution containing 0.05% Triton X100, 0.5% NDS and the primary antibodies rabbit anti‐GFP (1:1000, Abcam), and chicken anti‐vimentin (1:4000, Abcam). Following this incubation, slices were washed in PBS and transferred to PBS solution of secondary antibodies for 24 hours at 4°C; donkey anti‐rabbit Cy5 (1:800, Jackson ImmunoResearch) and donkey anti‐chicken Alexa 488 (1:800, Abcam). The secondary antibodies were then washed off in PBS and the slices were mounted on gelatine‐coated glass slides using VectaShield medium (Vector Laboratories, USA). Slices were then imaged on a confocal microscope (Leica SP5 upright) under the 20x objective. Z‐stacks were acquired in 3 µm steps and viewed as maximal intensity projections.

Northeast R.C., Chrobok L., Hughes A.T., Petit C, & Piggins H.D. (2019). Keeping time in the lamina terminalis: Novel oscillator properties of forebrain sensory circumventricular organs. The FASEB Journal, 34(1), 974-987.

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Yeast Total Extract Preparation

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Yeast total extracts were prepared as previously described (Knop et al., 1999 (link)). 1.5 × 10⁸ cells from OD₆₀₀ = 0.4–0.6 cultures were resuspended in 1150 µl lysis buffer (0.24 M NaOH, 1% β-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 5 µM Pepstatin A, 10 µM Leupeptin). After incubation on ice for 20 min, 150 µl 55% trichloroacetic acid (TCA) was added to precipitate proteins on ice for 20 min. The mixture was centrifuged at 16100 rpm at 4°C for 10 min. The pellet was resuspened in 250 µl HU buffer (8 M urea, 5% SDS, 200 mM Tris-HCl [pH 6.8], 1 mM EDTA, 5% β-mercaptoethanol, and 1% bromophenol blue) and incubated at 65°C for 10 min, followed by 16100 rpm centrifugation at RT for 5 min. The supernatant was used for subsequent analyses. Immunoblotting was performed with primary antibodies: rabbit-anti-GFP (1:2000) (Abcam), and mouse-anti-PGK1 (1:1000) (Abcam). Mouse-anti-rabbit-IgG (1:10,000) (Santa Cruz) and goat-anti-mouse-IgG (1:10,000) (Santa Cruz) were used as secondary antibodies.

Xing W., Muhlrad D., Parker R, & Rosen M.K. (2020). A quantitative inventory of yeast P body proteins reveals principles of composition and specificity. eLife, 9, e56525.

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Visualizing DNA Damage Markers in U2OS Cells

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For staining lacO tags, lentivirally infected U2OS-lacO/GFPLacI cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature (RT), permeabilized in 0.5% NP40/PBS buffer for 10 min at RT, blocked in 1% bovine serum albumin (BSA) in PBS, and incubated with rabbit anti-GFP (1:500) (Abcam, 290). For staining RAD51 foci, lentivirally infected U2OS cells were permeabilized in Triton X-100 buffer (0.5% Triton X-100, 20 mM Hepes-KOH at pH 7.9, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 5 min at RT and fixed in 3% paraformaldehyde (in PBS, 2% sucrose) for 10 min at RT, followed by permeabilization in Triton X-100 buffer for 10 min RT. Cells were blocked in 1% BSA/PBS, followed by incubation with rabbit anti-RAD51 (4 μg/ml) (Santa Cruz Biotechnology, 8349). Primary antibodies were detected with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibodies (1:100) (Jackson Laboratories). DNA was stained with DAPI (0.2 μg/ml) to score micronuclei.

Ramamoorthy M, & Smith S. (2015). Loss of ATRX suppresses resolution of telomere cohesion to control recombination in ALT cancer cells. Cancer cell, 28(3), 357-369.

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Immunogold Labeling and EM Analysis of GFP-LC3

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Immunogold labeling and EM analysis of GFP‐LC3 stable U2OS cells transiently transfected with HA‐Rab7b were performed as described in 44. For double labeling, sections were first labeled with rabbit anti‐GFP (Abcam) at 1:100 and protein A gold 5 nm (Cell Microscopy Center, UMC) at 1:50, followed by anti‐HA (Biolegend) at 1:50 and protein A gold 10 nm (Cell Microscopy Center, UMC) at 1:50. Sections were analyzed with a JEM‐1400 TEM microscope (Jeol), and images were recorded with TemCam‐F216 camera using EM MENU software (both from Tvips).

Kjos I., Borg Distefano M., Sætre F., Repnik U., Holland P., Jones A.T., Engedal N., Simonsen A., Bakke O, & Progida C. (2017). Rab7b modulates autophagic flux by interacting with Atg4B. EMBO Reports, 18(10), 1727-1739.

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Spleen Immunofluorescence Imaging Protocol

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Spleens were frozen in OCT. Sections (10μm) and blocked with 5% goat serum for 1h. Rabbit anti-GFP (Abcam Plc. Cambridge MA), anti-rabbit Alexa-555, anti-IgM-Alexa488 or Alexa647, anti-B220-biotin, streptavidin-Alexa488 (or Alexa647), anti-CD3-Alexa647 (Thermo Fisher Scientific) and anti-SIGNR-1-Alexa488 (eBioscience Inc) were used as indicated. Whole spleen images were acquired with Nikon 90i and A1 immunofluorescence or confocal microscopes connected to a motorized stage. Analysis was carried out with Nikon NIS Elements software.

Mohib K., Cherukuri A., Zhou Y., Ding Q., Watkins S.C, & Rothstein D.M. (2019). Antigen-dependent interactions between regulatory B cells and T cells at the T:B border inhibit subsequent T cell interactions with DCs. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(1), 52-63.

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Standard and Whole Mount Immunostaining

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Standard and whole mount immunostaining procedures were performed. Tissue sections on glass slides were fixed in 4% paraformaldehyde for 15 minutes before incubation in 10% serum in 0.1% PBT (0.1% Triton X-100 in PBS) for 1 hour and then in primary antibody (in 5% serum/0.1% PBT) overnight at 4°C. The primary antibodies used were: rat anti-K8 (1:50, University of Iowa), rabbit anti-K17 (1:200, Epitomics), chicken anti-GFP (1:1000, Abcam), rabbit anti-GFP (1:500, Abcam), rabbit anti-Sox2 (1:500, Stemgent), and rabbit anti-NCAM (1:500, Millipore). Alexa Fluor-conjugated secondary antibodies (1:2000, Invitrogen) were used to detect the signals. Whole mount immunostaining followed the online protocol as described [52 ]. Concomitant staining of littermate control tissue and control staining where the primary antibody was omitted were used to confirm the specificity of experimental staining. Confocal images were acquired with the Zeiss LSM 710 Confocal system (Carl Zeiss Inc, Thornwood, NY).
The Troma-1/K8 antibody developed by Dr. Philippe Brulet and Dr. Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

Xiao Y., Thoresen D.T., Miao L., Williams J.S., Wang C., Atit R.P., Wong S.Y, & Brownell I. (2016). A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development. PLoS Genetics, 12(7), e1006150.

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Protein Extraction and Western Blotting

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Cells were harvested and lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na^{3 (link)}VO^{4 (link)}, 2 mM EDTA, 1mM PMSF and protease inhibitors (cat. no. HY-K0010; MedChemExpress)) as previously described.^{30 (link)} Briefly, samples were loaded and separated by SDSPAGE, transferred to PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk (BD Bioscience) at room temperature for 1 h. Subsequently, membranes were probed overnight at 4°C with the following primary antibodies: mouse anti-spastin (1:100, cat. no. sc-374068; Santa Cruz Biotechnology, Inc.), mouse anti-GAPDH (1:5,000, cat. no. ab8245; Abcam), rabbit anti-GFP (1:1,000, cat. no. ab290; Abcam), rabbit anti-mCherry (1:1,000, cat. no. ab125096; Abcam), mouse anti-Flag (1:1,000, cat. no. F1802; Sigma-Aldrich; Merck KGaA). HRP-conjugated secondary antibodies (cat. nos. AS038 and AS003; ABclonal Biotech Co, Ltd.) were used for protein signal detection with an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

Zou J., Cai Z., Liang Z., Liang Y., Zhang G., Yang J., Zhang Y., Lin H, & Tan M. (2021). Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability. European Journal of Histochemistry : EJH, 65(4), 3352.

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Immunohistochemistry of Mouse Brain Sections

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Following the final drinking session, mice were deeply anesthetized using a mixture of ketamine (250 mg/kg) and xylazine (25.0 mg/kg). Cardiac perfusions were performed using phosphate buffered saline (PBS) and then 4% paraformaldehyde (PFA) in PBS. Brains were extracted and placed into 4% PFA (in PBS) for 48 hours before being submerged in cryoprotectant (30% glycerol in PBS). Brains were sectioned into 30 μm sections on a freezing microtome (Leica, Wetzlar, Germany) and stored in 0.01% sodium azide (in PBS). Immunohistochemistry was performed using rabbit anti-GFP (1:20,000; Abcam, Cambridge, MA, cat# ab290) with goat anti-rabbit IgG Alexa Fluor 488 (1:500; Invitrogen, Carlsbad, CA) for NAc containing sections, and rabbit anti-mCherry (1:500; Abcam) primary antibodies and goat anti-rabbit IgG Alexa Fluor 594 (1:500; Invitrogen) secondary antibodies for CeA containing sections. Sections were counterstained and mounted using Vectashield with DAPI (Vector Labs, Burlingame, CA). Viral infusions were verified using a fluorescent microscope (15 mice were excluded from final analysis due to misplaced viral expression).

Borrego M.B., Grigsby K.B., Townsley K.G., Chan A., Firsick E.F., Tran A, & Ozburn A.R. (2021). Central nucleus of the amygdala projections onto the nucleus accumbens core regulate binge-like alcohol drinking in a CRF-dependent manner. Neuropharmacology, 203, 108874.

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