Cells were fixed with 2% formaldehyde, permeated with 0,05% Triton X-100, blocked 1h with 5% BSA, and subjected to staining using anti-lamin A (Santa Cruz), anti-NF-YA monoclonal (Santa Cruz), Cy3-conjugated donkey anti-mouse and Cy2-conjugated donkey anti-rabbit (Jackson Immuno Research Laboratories). Slides were analyzed within 24 h. As control, single immunofluorescence labeling for each antibody, and immunofluorescence labeling without primary antibody was performed (data not shown). All experiments were performed several times with similar results. Images were recorded by using a Zeiss LSM 510 Meta confocal laser scanning microscope equipped with a 20X and 60X/1.23 NA oil immersion objective. As laser (488 and 514 nm), and HeNe laser (543 nm) were used to excite the fluorophores. The LSM 510 R. 3.2 META (Zeiss) image analysis software was used.
Cy2 conjugated donkey anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United States
Cy2-conjugated donkey anti-rabbit is a secondary antibody that is conjugated to the Cy2 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Fv100, by Olympus (4 mentions)
Cy2 conjugated donkey anti mouse, by Jackson ImmunoResearch (4 mentions)
Mouse anti o4, by Merck Group (4 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (3 mentions)
Cy3 conjugated donkey anti rabbit, by Jackson ImmunoResearch (2 mentions)
Mouse anti map2, by Merck Group (2 mentions)
Lsm 710 confocal microscope, by Zeiss (2 mentions)
Anti lamin a, by Santa Cruz Biotechnology (1 mentions)
Cy3 conjugated donkey anti mouse, by Jackson ImmunoResearch (1 mentions)
Lsm 510 meta confocal laser scanning microscope, by Zeiss (1 mentions)
Anti nf ya monoclonal, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Rabbit α mafa, by Fortis Life Sciences (1 mentions)
Rabbit α mafb, by Fortis Life Sciences (1 mentions)
Cy5 conjugated donkey anti guinea pig, by Jackson ImmunoResearch (1 mentions)
Biotin conjugated donkey anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
13 protocols using cy2 conjugated donkey anti rabbit
1
Immunofluorescence Labeling of Lamin A and NF-YA
2
Dual Labeling for Cholinergic Neurons
3
Immunofluorescent Labeling of Pancreatic Sections
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The conditions used for paraffin embedding pancreatic sections and immunofluorescence labeling were described previously (20 (link)). The following primary antibodies were used for immunostaining: guinea pig α-insulin (1:2,000; Linco Research), guinea pig α-glucagon (1:2,000; Linco Research), rabbit α-glucagon (1:2,000; Linco Research), rabbit α-MafB (1:10,000; Bethyl Laboratories), rabbit α-MafA (1:1,000, Bethyl Laboratories), rabbit α-Slc30a8 (1:1,000, Mellitech), and rabbit α-Glut2 (1:1,000; Chemicon). Species-matched secondary antibodies were used for immune detection (Cy5-conjugated donkey anti–guinea pig, Cy5-conjugated donkey anti-sheep, Cy2-conjugated donkey anti–guinea pig, Cy2-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-rabbit [all 1:500; Jackson ImmunoResearch Laboratories]). Cy3-conjugated tyramide signal amplification (1:400, PerkinElmer) was used for detecting MafB labeling with the biotin-conjugated donkey anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch Laboratories). Nuclear counterstaining was performed using YoPro1 or DAPI (Invitrogen). Immunofluorescence images were acquired with a Carl Zeiss LSM510 confocal microscope.
4
Immunofluorescence Analysis of Hippocampal Neuron Morphology
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Hippocampal neurons grown 1 day in vitro (DIV) were treated with 5 µM of PBP10 or 7.5 µM of WRW4 for 3 days. Control samples were treated with vehicle control (water). Cells were fixed with 3.7% paraformaldehyde (Sigma) at the end of 3 days and permeabilised with 0.3% Triton-X (Sigma). The fixed samples were then rinsed with PBS and blocked with 10% normal goat serum (Merck) at room temperature for 30 to 45 min. Cells were incubated with primary antibodies against MAP2 (polyclonal rabbit anti-MAP2, Synaptic Systems, dilution 1:400) and Tau (polyclonal guinea pig anti-Tau, Synaptic Systems, dilution 1:400) at room temperature for 1 h before three consecutive washes with PBS to remove excess antibodies. Following this, cells were incubated with secondary antibodies (Cy2-conjugated donkey anti-rabbit, dilution 1:200; Cy3-conjugated donkey anti-guinea pig, dilution 1:400; both from Jackson ImmunoResearch) for 1 h at room temperature. Cells were further washed with PBS prior to mounting onto glass slides with Fluoro-Gel II with DAPI (Electron Microscopy Sciences).
5
Immunocytochemistry of Neurons
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Coverslips with neurons were fixed with 4% paraformaldehyde/4% sucrose on ice, washed, permeabilized with 0.1% triton X-100 for 5 min at room temperature, washed, blocked for 30 min at room temperature with 5% BSA/5% normal donkey serum and then incubated overnight at 4°C with primary antibody (rabbit anti-MAP-2 at 1∶500; EMD Millipore, Billerica, MA, USA). Coverslips were then washed, incubated with secondary antibody (Cy2-conjugated donkey anti-rabbit; Jackson ImmunoResearch, West Grove, PA, USA) for 1 hr at room temperature, washed and incubated with DAPI and mounted on slides with Fluoromount-G (Southern Biotech, Birmingham, AL, USA).
6
Immunofluorescence Labeling of Paraffin Tissue
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For immunofluorescence labeling using paraffin embedded tissue sections the treatment procedure was performed as previously described8 (link),22 (link),88 (link). In sum, after deparaffinizing and buffering, the slides were incubated with a mixture of the rabbit IL-6 antibody (see above) and mouse anti-MAP2 (microtubule-associated protein 2, 1:200; Millipore, Temecula, USA) as well as mouse anti-GFAP (1:1000; Sigma-Aldrich, St. Louis, USA) in 5% fetal calf serum in buffer over night at 4 °C. The primary antibodies were visualized with Cy2-conjugated donkey anti-rabbit (1:400) and Cy5-conjugated donkey anti-mouse IgG (1:200; both Jackson ImmunoResearch, West Grove, USA) after 2 h incubation. Further, slides were stained with Hoechst 33342 (1:1000, Molecular Probes, Leiden, Netherlands) to identify the cell nuclei by auto-fluorescence utilizing an ultraviolet laser (362 nm). Strict internal control runs were done without primary antibodies.
The immunofluorescence was investigated by a confocal laser scanning microscope (Leica SP8 confocal microscope; Leica, Wetzlar, Germany) using excitation wavelengths of 488 nm (argon laser, yellow-green Cy2-immunofluorescence labelling), 543 nm (helium/neon1, red Cy3-immunofluorescence) and 633 nm (helium/neon2, blue Cy5-labelling).
The immunofluorescence was investigated by a confocal laser scanning microscope (Leica SP8 confocal microscope; Leica, Wetzlar, Germany) using excitation wavelengths of 488 nm (argon laser, yellow-green Cy2-immunofluorescence labelling), 543 nm (helium/neon1, red Cy3-immunofluorescence) and 633 nm (helium/neon2, blue Cy5-labelling).
7
Immunofluorescence Staining of Neural Cells
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Cells were fixed in 4% paraformaldehyde (PFA) for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Rabbit anti-HMGB1 (1:500, Abcam, Cambridge, UK), mouse anti-MAP2 (1:1,000, Sigma, St. Louis, MO), rabbit anti-GFAP (1:500, Sigma) and mouse anti-O4 (1:100, Sigma) were used as primary antibodies. Secondary antibodies included Cy2-conjugated donkey anti-mouse (1:500) and Cy2-conjugated donkey anti-rabbit (1:500, Jackson ImmunoResearch, West Grove, PA). Samples were examined under a fluorescence microscope (FV-100, Olympus, Japan).
8
Immunocytochemical Characterization of Cell Cultures
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Cells cultured on coverslips were fixed in 4% paraformaldehyde (PFA) for 10 min and rinsed with PBS for three times, followed by permeabilization with 0.2% Triton X-100 for 10 min. Samples were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with primary antibodies overnight at 4 °C. Cells were then incubated with Cy2-conjugated secondary antibodies for 1 h. Washing with PBS was performed between each staining step. Nuclei were counter-stained with Hoechst for 5 min. The antibodies used included mouse anti-mitogen associated protein 2 (MAP2, 1:1000, Sigma, St. Louis, MO), rabbit anti-gial fibrilling acidic protein (GFAP, 1:500, Sigma), mouse anti-O4 (1:100, Sigma), Cy2-conjugated donkey anti-mouse (1:500) and Cy2-conjugated donkey anti-rabbit (1:500, Jackson ImmunoResearch, West Grove, PA). Samples were examined under a fluorescence microscope (FV-100, Olympus, Japan).
9
Drosophila Brain Immunohistochemistry
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Adult fly heads were fixed with 4% p-formaldehyde (pH 7.5) for 30–40 min at room temperature. Brains were dissected and rinsed four times in PT buffer (PBS with 0.1% Triton X-100) for 30 min. Samples were blocked in 7% normal goat serum (in PT) for 1 h, and incubated with primary antibodies at room temperature for 2 days. The primary antibodies employed were chicken anti-GFP 1:500 (Aves Labs, Inc, USA), rabbit anti-DsRed 1:500 (Clontech, USA) and homemade rat anti-Drosophila-PDF 1:500 (Depetris-Chauvin et al., 2011 (link)). Samples were washed 4 x 15 min in PT, and incubated with secondary antibody at 1:250 for 2 h at room temperature. Secondary antibodies were washed 4 x 15 min in PT and mounted in Vectashield antifade mounting medium (Vector Laboratories, USA). The secondary antibodies used were Cy2-conjugated donkey anti-rabbit, Alexa Fluor 647-conjugated AffiniPure donkey anti-rat and Cy3-conjugated AffiniPure donkey anti-rabbit (Jackson ImmunoResearch, USA). Images were taken on a Zeiss LSM 710 confocal microscope.
10
Immunohistochemical Staining of PDGFRβ
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A subset of sections was washed in PBS 0.01 M for 10 min, followed by two times in 0.1%TritonX100 in PBS (PBST) for 5 min. The PBST was removed and blocking (10%normal donkey serum (NDS), 1%bovine serum albumin (BSA), 0.5 %Triton X100 in PBS) was performed for 1 h at room temperature. This was directly followed by incubation overnight at room temperature with primary antibody (PDGFRβ; host: rabbit; diluted 1:100; Abcam; cat# ab32570) diluted in antibody solution (ABS; 3%NDS, 1 %BSA, 0.1%Triton X100 in PBS). The following day the sections were rinsed in 0.1%PBST two times for 1 min and three times for 10 min. Secondary antibody (CY2-conjugated donkey anti-rabbit; diluted 1:500; Jackson ImmunoResearch Labs.; cat# 711-225-152) was diluted in ABS, and the sections were incubated for 1 h at room temperature. After the second incubation, the sections were washed in PBS for 10 min, three times. All sections were transferred to ddH2O and mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific; cat# P36934). Z-stack images were acquired using a Zeiss LSM 710 confocal microscope, at 20×, 40×, or 63×magnification at an optical thickness of 1μm. The stacks obtained were 3D rendered using ImageJ software, FIJI [36 (link)]. The images were used for qualitative purposes.
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