The following antibodies were used in the present study: rat monoclonal anti-myelin basic protein (MBP; Cat. #: MAB386; Millipore, Billerica, MA); rabbit anti-parvalbumin (PV; Cat. #: 195002; Synaptic Systems, Gottinggen, Germany); rabbit anti-degraded MBP (dMBP; Cat. #: AB5864; Millipore, Billerica, MA); Cy3-, and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). All antibodies were used in a 1:200 dilution. The nuclear dye Hoechst 33342 was purchased from Invitrogen (Cat. #: H3570; Carlsbad, CA, USA).
Cy3 and cy5 conjugated secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom
Cy3- and Cy5-conjugated secondary antibodies are fluorescent-labeled antibodies that can be used to detect and visualize specific target proteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry. These conjugated antibodies provide a sensitive and reliable way to amplify and detect the signal from primary antibodies, enabling researchers to study protein expression and localization.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti lamin b1, by Merck Group (1 mentions)
Alexa fluor 633 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Rhodamine conjugated phalloidin, by Merck Group (1 mentions)
Anti at8, by Thermo Fisher Scientific (1 mentions)
Anti at100, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Anti gfp, by Abcam (1 mentions)
Rabbit anti 53bp1, by Fortis Life Sciences (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
T8660, by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Anti β actin ac 74, by Merck Group (1 mentions)
Ab124131, by Abcam (1 mentions)
Ccd spot camera, by Nikon (1 mentions)
10 protocols using cy3 and cy5 conjugated secondary antibodies
1
Immunofluorescent Staining of Brain Markers
2
Whole-mount Fly Eye and Brain Imaging
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All flies were age-matched for experiments. The whole-mount preparation of fly eyes and brains was performed as previously described46 (link).The following primary antibodies were used with the indicated dilutions: anti-lamin B1 (Sigma, 1:20), anti-GABA (GeneTex, 1:200), anti-human pan-tau (Dako, 1:200), anti-tau-C3 (Invitrogen, 1:200), anti-GFP (Abcam, 1:100), anti-AT8 (Thermo, 1:200), anti-AT100 (Thermo, 1:100), and anti-PHF1 (Abcam, 1:100). Alexa Fluor 488, Cy3, and Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 1:100 dilutions. F-Actin enriched rhabdomere and spots of aberrant actin accumulations were labeled by rhodamine-conjugated phalloidin (Sigma, 1:20) and Alexa Fluor 633-conjugated phalloidin (Thermo, 1:50), respectively. Samples were analyzed on Zeiss LSM510 or LSM800 confocal microscopes.
3
Immunofluorescent Detection of DNA Damage Foci
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P133 cells were fixed in 3.7% formaldehyde/PBS for 10 min, permeabilized with 0.5% Triton/PBS, and blocked with 10% FCS/PBS. The primary antibodies used were mouse anti-γH2AX (Upstate Biotechnology) and rabbit anti-53BP1 (Bethyl Laboratories). Appropriate Cy3- and Cy5-conjugated secondary antibodies were added (Jackson ImmunoResearch Laboratories). Images were taken with a Bio-Rad confocal microscope, and analysis was performed using ImageJ. Colocalization of both signals was considered as a DSB foci. Foci analysis was performed from at least 45 nuclei for each condition.
4
Hepatitis B Virus Antigen Detection
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The monoclonal MA18/7 anti-PreS1 domain of LHBs was kindly provided by Dr. Dieter Glebe, Giessen [3 (link),19 (link)]. The polyclonal rabbit against denatured HBV core K46 [20 (link)] was a gift from Dr. Reinhild Prange, Department of Medical Microbiology and Hygiene, Johannes Gutenberg-Universität Mainz, Mainz, Germany. Polyclonal rabbit anti-HBV core B0586 was purchased from Dako, Denmark. Anti-beta-Actin was ordered from Sigma-Aldrich (St. Louis, MO, USA). For immunofluorescence staining, Alexa Fluor 488-conjugated (Invitrogen), Cy3- and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc., Ely, UK) were used. For quantification of HBcAg, HBsAg and HBeAg, a QuickTiterTM Hepatitis B Core Antigen (HBcAg) ELISA Kit (Cell Biolabs, INC, San Diego, CA, USA), and an Enzygnost® HBsAg 6.0 and HBeAg (ELISA; Siemens, Munich, Germany) were ordered. The antobodies were generated by Seramun Institute (Heidesee, Germany). The ethic approval umber is from the RP Potsdam: 2347-A-15-1-2018.
5
Intravitreal Cell Injection Analysis
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Eight months after the intravitreal cell injections, mice were sacrificed and eyes were fixed for 1 h in 4% PA. Lenses with attached NS cells were removed and stained with goat anti-GDNF and rabbit anti-CNTF antibodies, to analyze the expression of both growth factors, or with rabbit anti-glial fibrillary acidic protein (GFAP; Dako Cytomation GmbH, Hamburg, Germany; Z0334; 1:500) and mouse anti-β-tubulin III (Sigma-Aldrich; T8660; 1:1000) antibodies, to monitor the differentiation of the grafted NS cells. Primary antibodies were detected with Cy3- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Inc.).
6
Antibody Panel for Protein Analysis
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The following commercial antibodies were used: anti-transferrin-receptor (H68.4, Thermo Scientific), anti-α-taxilin (H-66, Santa Cruz Biotechnology), anti-α-taxilin (atlas antibodies, Sigma), anti-HCV-NS3 (8G-2, Abcam), anti-LAMP-2/Cd107b (AF6228, R&D Systems), anti-EGFR (EP38Y, abcam), anti-LDLR (Thermo Scientific), and anti-β-actin (AC-74, Sigma). For the recycling kinetic anti-TfR (TFRC/1818, Biozol) was used. For detection of HCV NS5A, a polyclonal rabbit-derived serum was used (Bürckstümmer et al., 2006 (link)). IRDye® 800CW and IRDye® 680RD secondary antibodies for Western blotting were obtained from LI-COR. For immunofluorescence staining, Alexa Fluor 488-conjugated secondary antibodies (Invitrogen) as well as Cy3- and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc.) were used.
7
Immunohistochemistry Staining of Sciatic Nerve and DRG
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Immunohistochemistry staining was conducted as previously described [9 (link)]. In brief, after anesthetizing the mice with isoflurane, the animals were perfused with saline followed by 4% PFA. The sciatic nerves and the DRG were removed and post-fixed in 4% PFA for 8–12 h. The sciatic nerve sections and DRG slices (12 μm) were sectioned using a cryostat machine followed by immunohistochemistry staining. After washing, the slices were permeabilized with 0.3% Triton X-100 for 10 min, and blocked with 10% BSA for 2 h at room temperature, and processed for immunostaining with the following primary antibodies: S100 β (1:400, mouse; Sigma, AMAB91038), NF200 (1:300, mouse; Sigma, N0142), Panx1 (1:200, rabbit; Abcam, ab124131) overnight at 4 °C. Then, the slices were incubated with a mixture of cy3 and 488-, or cy3 and cy5-conjugated secondary antibodies (1:1000; Jackson Immuno Research) for 2 h at room temperature. For the staining of neurons in the DRG slices, Nissl staining (1:200, Thermo, N-21483) was applied and performed via the manufacturer’s instructions. Stained samples were examined using a fluorescence microscope (Nikon), and images were captured with a CCD Spot camera.
8
Indirect Immunofluorescence Staining Protocol
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For indirect immunofluorescence staining, cells were seeded on Nunc™ Lab-Tek™ II CC2™ chamber slides from Thermo Fisher Scientific (Schwerte). Briefly, cells were fixed for 15 min with 4% PFA and permeabilized for 5 min with 0.1% Triton™ X-100 at room temperature. The following primary antibodies were used for staining: polyclonal rabbit antibody against pericentrin (abcam®, Cambridge, UK), monoclonal mouse antibody against FITC-conjugated α-tubulin (Sigma-Aldrich) and human immune serum against centromere (anti-centromere antibody, ACA, ImmunoVision, Springdale, USA). Cy3 and Cy5-conjugated secondary antibodies were obtained from Jackson Immunoresearch (Newmarket, UK). DNA was stained using DAPI (4′,6-diamidino-2-phenylindole-dihydrochloride, Roche). Slides were examined using an AxioObserver.Z1 microscope with a HCX PL APO CS 63.0×1.4 oil UV objective (Zeiss, Göttingen) and images were taken using a confocal laser scanning microscope (CLSM, Leica CTR 6500, Heidelberg).
9
Immunostaining of Neuronal Synapses
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Example 5
Neurons were fixed and stained for synaptic markers at 11 DIV. Neuronal media was replaced with 1×PBS and neurons were then fixed with 4% paraformaldehyde/4% sucrose for 8 min at room temperature. Coverslips were then washed three times with 1×PBS for 5 minutes each and incubated overnight at 4° C. in a humidified chamber with primary antibody. All antibody dilutions were prepared in 1×GDB (0.1% gelatin, 0.3% TritonX-100, 4.2% 0.4M phosphate buffer, and 9% 5 M NaCl). After overnight incubation, coverslips were washed three times with 1×PBS for 5 minutes each and then incubated with appropriate Cy3- and Cy5-conjugated secondary antibodies (1:500 each; Jackson ImmunoResearch Laboratories) in 1×GDB for 2 hours at room temperature. Coverslips were then washed three times with 1×PBS for 10 minutes each, dipped in dH2O, and mounted on glass slides with Aquamount (Lerner Laboratories). The following antibodies were used: mouse (ms) α-GAD65 (1:1000, lipore), rabbit (rb) α-GA BAAR γ2 (1:100, Millipore), ms α-Gephyrin (1:500, Synaptic Systems), rb α-Synapsin I (1:1000, Millipore), ms α-GluA2 (1:500, NeuroMab), ms α-MAP2 (1:1000, Sigma), α-ms 488 (1:1000, Invitrogen), α-ms Cy3 (1:500, Jackson Immuno), α-rb Cy5 (1:500, JacksonImmuno).
10
Immunofluorescence Imaging of STARD3 and Lysosome Localization
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Cells were grown on glass coverslips, treated or not with 1 μg/ml of U18666A ((3β)‐3‐{2‐(Diethylamino)ethoxy}androst‐5‐en‐17‐one, 662015 Calbiochem) during 1 h, fixed 10 min at RT with 4% paraformaldehyde (PFA) in phosphate‐buffered saline (PBS), and permeabilized in a nitrogen bath for 30 s. After blocking in 1% BSA in PBS, cells were incubated at RT with GFP‐D4 solution in PBS for 1 h. Then, cells were rapidly washed and re‐fixed with PFA before a second blocking step. Primary antibodies, rabbit anti‐STARD3 (1:1,000; Alpy et al, 2001 ) and mouse anti‐Lamp1 H4A3 (1:50; DSHB), were incubated overnight at 4°C, and after three washes in PBS, cells were incubated for 1 h with Cy3‐ and Cy5‐conjugated secondary antibodies (Jackson ImmunoResearch). After two washes, nuclei were counterstained with Hoescht‐33258 dye and slides were mounted in ProLong Gold (Invitrogen). Observations were made with confocal microscope (Leica SP8 UV, 63×, NA 1.4).
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