RBPs-transfected neurons were co-transfected with the pEGFP-N1 plasmid to visualize dendritic spines with GFP. They were fixed at 11 DIV for FUSΔNLS-transfected neurons, or 10 DIV for TDP-43G348C-transfected neurons, with 3.7% formaldehyde in PBS for 10 min. After washing with PBS, the neurons were treated with 0.5% Triton X-100 in PBS for 10 min. After washing with PBS and blocking with 10% FBS in DMEM, presynapses were labeled with an anti-synapsin I antibody (Merck Millipore) and a Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch). After washing with PBS, the specimens were mounted in Mowiol. Fluorescence images were acquired using the IX83 inverted microscope with the PlanApo 60 × oil objective and the Prime 4.2 Megapixel sCMOS camera. The images were analyzed using ImageJ. Dendritic protrusions where synapsin I was attached were deemed to be spines. The number of spines was counted along the dendrites and normalized by the length of the dendrites. The mushroom spine was defined as having a maximum head width/neck width ratio of 1.5 or greater, which is often used as a cutoff value [80 (link),81 (link)].
Cy3 conjugated anti rabbit igg antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated anti-rabbit IgG antibody is a secondary antibody that is used to detect the presence of rabbit primary antibodies in various immunoassays and imaging applications. The antibody is conjugated to the fluorescent dye Cy3, which allows for the detection of the bound rabbit primary antibody using appropriate instrumentation.
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Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Psinrep5, by Thermo Fisher Scientific (2 mentions)
Anti glua1 n, by Alomone (2 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Anisomycin, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp linked anti rabbit immunoglobulin g igg, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Primary rabbit antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Cm3050, by Leica (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Hematoxylin and eosin h e, by Muto Pure Chemicals (1 mentions)
6 protocols using cy3 conjugated anti rabbit igg antibody
1
Visualizing Neuronal Spine Morphology
2
Sindbis Virus Expression of PTPN11 in Neurons
3
Sindbis Virus Expression of PTPN11 in Neurons
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The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into a Sindbis viral expression vector (pSinRep5; Invitrogen) and GFP was inserted into the 3′ region of the coding sequence along with an additional subgenomic promoter for bicistronic expression. Sindbis viruses were produced according to the manufacturer’s protocol (Invitrogen) and directly added to the medium of cultured rat hippocampal neurons (DIV21). Twelve hours after infection, immunocytochemistry was performed with or without permeabilization by using anti-GluA1-N (#AGC-004, Alomone labs) antibody and Cy3–conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Lab). Images were acquired by using confocal microscope (Zeiss LSM 710) and analyzed by using ImageJ (ver. 1.42q).
4
Spinal Cord Immunofluorescence Imaging
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Immunofluorescence was performed as described previously [21 (link)]. For human spinal cords, 20 μm sections were made of fresh froZen tissue, air dried on slides, and fixed in ice-cold acetone for 10 min. In brief, after blocking for 1 h, the sections were incubated with primary antibodies overnight at 4 °C. Bound antibodies were detected with appropriate Alexa Fluor-conjugated anti-rabbit, mouse, rat, goat, or guinea pig IgG antibodies (Thermo Fisher Scientific). For detecting ErbB3 antibody, Cy3-conjugated anti-rabbit IgG antibody (Jackson Laboratories, USA) was used. Immunostained images were obtained by confocal laser scanning microscopy (LSM 5 Exciter, LSM-700; Carl Zeiss AG, Germany) and the equipped software (Zen; Carl Zeiss AG).
5
Investigating JNK Signaling Pathway
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Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-Jun N-terminal kinase (JNK), phospho-c-Jun N-terminal kinase (p-JNK: Thr183/Tyr185), E-cadherin, ×10 cell lysis buffer, and horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG) were purchased from Cell Signaling Technology (Waltham, MA, USA). Fluorescein isothiocyanate-labeled phalloidin (FITC-phalloidin), SP600125 (a JNK inhibitor), anisomycin (a JNK activator), puromycin, and 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cy3-conjugated anti-rabbit IgG antibody was obtained from Jackson ImmunoResearch (West Grove, PA, USA). The JNK small hairpin RNA (shRNA) (shJNK) plasmid was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lipofectamine LTX and Plus reagents were obtained from Invitrogen (Carlsbad, CA, USA). psPAX2, a virus packaging vector, and pMD2.G, an envelope protein vector, were gifts from Dr. Zang-Hee Lee (Seoul National University, Seoul, Korea). Y-27632 (Tocris Cookson, Avonmouth, UK) was used to inhibit the activity of Rho-associated kinase (ROCK). Gibco 0.25% trypsin-EDTA was obtained from Fisher Scientific (Pittsburgh, PA, USA).
6
Retinal Ganglion Cell Labeling in Rotenone-Induced Retinal Degeneration
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Eyes were dissected from SD rats seven days after intravitreal injection of rotenone alone or together with Ntf5 recombinant proteins and were fixed with 4% paraformaldehyde in PBS overnight at 4 °C. Fixed eyes were embedded in optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan) and were cut into 10 µm-thick sections using a cryostat CM3050S (Leica Biosystems, Wetzlar, Germany). Immunostaining of retinal sections was performed as reported before69 (link). Briefly, retinal sections were treated with blocking solution (10% normal donkey serum in PBS containing 0.01% Tween 20), followed by treatment with rabbit primary antibody (Abcam, Cambridge, UK; 1:500 dilutions with blocking solution) for RNA-binding protein with multiple splicing (RBPMS), which is a selective marker for retinal ganglion cells70 (link). After extensive washing, retinal sections were incubated with Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:500 dilutions with blocking solution) and 4′,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). To visualize the inner nuclear layer (INL) cells, retinal sections were stained with hematoxylin and eosin (HE; Muto Pure Chemicals, Tokyo, Japan).
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