Primary antibodies: Rabbit anti-cleaved Caspase 3 (9661S, Cell Signaling Technology, Danvers, USA), rabbit anti-Ki-67 (ab16667, Abcam, Cambridge, UK), rat anti-CD31 (DM3614P, Dianova, Hamburg, Germany), goat anti-CD32b (AF1460, R&D Systems, Minneapolis, USA), goat anti-Lyve1 (AF2125, R&D Systems, Minneapolis, USA), rat anti-Endomucin (14–5851–82, Thermo Fisher Scientific, Waltham, USA), rabbit anti-TRP-2 (ab74073, Abcam, Cambridge, UK), rabbit anti-wide spectrum cytokeratin (ab9377, Abcam, Cambridge, UK), goat anti-alpha smooth muscle actin (ab21027, abcam, Cambridge, UK), rat anti-CD45 (550539, BD Biosciences, Franklin Lakes, USA), rat anti-CD68 (137002, BioLegend, San Diego, USA), rabbit anti-Melan A (NBP1-30151, Novus, Minneapolis, USA). For Western blotting: rabbit anti-Akt (9272S, Cell Signaling Technology, Danvers, USA), rabbit anti-phospho-Akt (9271S, Cell Signaling Technology, Danvers, USA). Secondary antibodies: Alexa-Fluor 488, Alexa-Fluor 647 and Cy3-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany). For Western Blotting a rabbit anti-IgG HRP conjugated antibody was used. (Merck, Darmstadt, Germany).
Cy3 conjugated secondary antibody
Manufactured by Dianova
Sourced in Germany
The Cy3-conjugated secondary antibody is a fluorescent-labeled antibody that binds to the Fc region of primary antibodies. It is commonly used in various immunoassay techniques, such as immunofluorescence, flow cytometry, and Western blotting, to detect and visualize target proteins or cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Anti stat1 antibody c 24, by Santa Cruz Biotechnology (2 mentions)
Ab9377, by Abcam (1 mentions)
Ab21027, by Abcam (1 mentions)
Ab74073, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Goat anti lyve1 af2125, by R&D Systems (1 mentions)
Rat anti cd68, by BioLegend (1 mentions)
Rabbit anti akt 9272, by Cell Signaling Technology (1 mentions)
Alexa fluor 647, by Dianova (1 mentions)
Rat anti cd45, by BD (1 mentions)
Alexa fluor 488, by Dianova (1 mentions)
Estradiol, by Merck Group (1 mentions)
Alexa 633, by Thermo Fisher Scientific (1 mentions)
Insitupro machine, by Intavis (1 mentions)
Rat anti tubulin, by Abcam (1 mentions)
Estradiol, by Thermo Fisher Scientific (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Cd31 pecam 1, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
9 protocols using cy3 conjugated secondary antibody
1
Comprehensive Immunohistochemistry Panel
2
Immunofluorescence Imaging of Estradiol-Induced Cellular Processes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five days old seedlings were incubated in 1 ml liquid growth medium (0.5x MS medium, 1% sucrose, pH 5.8) containing 20 μM Estradiol (Sigma Aldrich) for 5-6h at plant room conditions in 24-well cell-culture plates. Incubation was stopped by fixation with 4% paraformaldehyde in MTSB. Immunofluorescence staining was performed as described [49 (link)] or with an InsituPro machine (Intavis) [50 (link)].
Antibodies used: rat anti-tubulin 1:600 (Abcam), rabbit anti-AtγCOP 1:1000 (Agrisera), rabbit anti-KNOLLE 1:2000 [49 (link)], rabbit anti-clathrin (1:600) [51 (link)]. Alexa633 (Invitrogen) or Cy3-conjugated secondary antibodies (Dianova) were diluted 1:600.
Live-cell imaging was performed with 2 μM FM4-64 (Invitrogen, Molecular Probes).
Estradiol induction was performed using 20μM Estradiol for 5-6h at plant room conditions in 24-well cell-culture plates.
Antibodies used: rat anti-tubulin 1:600 (Abcam), rabbit anti-AtγCOP 1:1000 (Agrisera), rabbit anti-KNOLLE 1:2000 [49 (link)], rabbit anti-clathrin (1:600) [51 (link)]. Alexa633 (Invitrogen) or Cy3-conjugated secondary antibodies (Dianova) were diluted 1:600.
Live-cell imaging was performed with 2 μM FM4-64 (Invitrogen, Molecular Probes).
Estradiol induction was performed using 20μM Estradiol for 5-6h at plant room conditions in 24-well cell-culture plates.
3
Immunohistochemical Analysis of Spinal Metastases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spine samples were transferred to a cryostat (Leica, Wetzlar, Germany), and cryosections were made at −20°C. Slides were dried and stored at −20 °C. For immunostaining, they were thawed and fixed in methanol (−20°C for 5 min) followed by 30 min incubation with blocking buffer (1% casein in PBS). The primary antibodies against Ki67 (Thermo Fisher Scientific, Waltham, USA) and CD31/PECAM-1 (BD Pharmingen, Franklin Lakes, USA) as well as FITC-conjugated and Cy3-conjugated secondary antibodies (Dianova, Hamburg, Germany) were added for 2 h, respectively. Cell nuclei staining was performed by incubation with 4',6-diamidino-2-phenylindole (1:100 in PBS, Roth, Karlsruhe, Germany) for 5 min. Slides were covered with Immu-Mount (Thermo Fisher Scientific, Waltham, USA). By fluorescence microscopy, 30 high-power fields from three different spinal metastases per group were randomly chosen, Ki67 positive and negative stained cell nuclei were compared by percentage with ImageJ software (NIH, Bethesda, USA), CD31 positive endothelial cells were counted and subsequently analyzed. Negative controls without processing primary antibodies did not display any specific immunoreactivity.
4
Immunofluorescence Staining of Tight Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coronal cryosections (10 µm in thickness) and cell monolayers were fixed with zinc-based fixative for 30 min and were permeabilized with 0.5% saponin in PBS for 15 min. Then, brain slices and cells were incubated for 30–60 min in blocking buffer consisting of 10% goat serum (Dianova, Hamburg, Germany) in PBST (0.1% Tween-20 in PBS) or 1% BSA in PBS, respectively. Subsequently, slices and cells were incubated overnight at 4°C with antibodies against occludin (1%; Invitrogen) or ZO-1 (1%; Invitrogen) followed by incubation with appropriate Cy3-conjugated secondary antibodies (0.5%; Dianova) for 1 h. All antibodies were diluted in LowCross-Buffer (Candor, Wangen, Germany). Slices and cells were incubated for 10 min with 0.02% DAPI (Invitrogen) in PBS to stain nuclei. Stained sections and cells on glass slides were then embedded in Mowiol mounting medium (Sigma-Aldrich, Steinheim, Germany). Fluorescence staining was recorded using an Olympus BX50 microscope with a Leica DC 500 camera. For confocal microscopy analysis, an Olympus IX81 confocal microscope and camera equipped with a 60×objective was used.
5
Immunocytochemical Characterization of Differentiated Neurons
6
3D Imaging of Xenopus Embryonic Hearts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optical projection tomography (OPT) was applied to obtain high-resolution three dimensional (3D) images of the heart in Xenopus embryos. Stage 43 embryos were fixed with Dent's solution (20% DMSO/80% methanol) and stained with the anti-CT3 antibody (1:50; DSHB, Iowa USA) and Cy3-conjugated secondary antibody 1:100 (Dianova, D). Xenopus embryos were mounted in 1% low melting agarose (Lonza, USA) and dehydrated overnight in 100% methanol at room temperature in the dark. Samples were cleared overnight at room temperature with benzylalcohol and benzylbenzoate (1:2) and imaged with an OPT Scanner 3001 M (Bioptonics Microscopy, UK). Pictures were taken with 1024 × 1024 pixel and 0.9° rotation step. The reconstruction of the data was performed using the NRecon software (SkyScan 3001). Imaris software 5.0 (Bitplane, Zurich, CH) was used to analyze OPT data.
7
STAT1 nuclear translocation in U3A cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear accumulation of recombinant untagged STAT1 was immunocytochemically detected in U3A cells expressing wild-type or mutant STAT1. Adherent cells grown on 8-well chamber slides were either left untreated or treated with IFNγ for 45 min. Interferon-prestimulated cells were then incubated in the presence of 500 nM staurosporine for an additional 0, 30 or 60 min and then fixed with methanol at −20°C for 20 min. After two washes in PBS, the cells were permeabilized with 1.0% Triton X-100 in PBS for 20 min and non-specific binding was blocked by incubation with 25% FCS/PBS for 45 min at RT. The samples were incubated for 45 min with anti-STAT1 antibody C-24 (Santa Cruz) diluted 1∶1000 in 25% FCS/PBS. After three washes in PBS, the specimens were incubated with Cy3-conjugated secondary antibody (Dianova), diluted 1∶500 in PBS, for 45 min at RT followed by nuclear staining with Hoechst dye. Finally, the samples were mounted and images were captured by fluorescence microscopy. Quantification was as described above.
8
Immunofluorescence Analysis of STAT1 in T. cruzi Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adherent HFF and U3A cells grown on 8-well chamber slides were either left uninfected or incubated with T. cruzi parasites for 18 h. Cells were fixed with methanol at −20°C for 20 min and, after two washes in PBS, permeabilized with 1.0% Triton X-100 in PBS for 20 min. Non-specific binding was blocked by incubation with 25% FCS/PBS for 45 min at RT before the samples were incubated for 45 min with anti-STAT1 antibody C-24 (Santa Cruz) diluted 1∶1000 in 25% FCS/PBS. After three washes in PBS, the specimens were incubated with Cy3-conjugated secondary antibody (Dianova), diluted 1∶500 in PBS, for an additional 45 min. Nuclei of human cells and intracellular parasites were detected by staining with Hoechst 33258 dye at a final concentration of 5 µg/ml. Samples were mounted in fluorescence mounting medium (Southern Biotech) and visualized using an Axiovert 200 M microscope (Carl Zeiss) equipped with appropriate fluorescence filters. Images were obtained with a CCD camera and further processed with the Image-Pro MDA5.1 (Media Cybernetics) software. For microscopic localization of GFP-tagged STAT1, cells were fixed with 4% paraformaldehyde in PBS and stained with Hoechst dye.
9
Corneal Hemangiogenesis and Lymphangiogenesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the assessment of inflammatory corneal hemangiogenesis and lymphangiogenesis, corneal suture placement was performed in WT, IL-10 À/À , Stat3 fl/fl , LysMCre Stat3 fl/fl , and WT treated with recombinant murine IL-10 or PBS. After the indicated time points, corneas (n Z 5 to 10 per group) were excised, fixed in acetone, blocked with 2% bovine serum albumin in PBS, and stained overnight with a LYVE-1 antibody (AngioBio, Del Mar, CA) and in indicated experiments with a fluorescein isothiocyanateeconjugated CD31 antibody (Acris Antibodies, Herford, Germany). On the next day, LYVE-1 was detected with a Cy3-conjugated secondary antibody (Dianova). Whole mount images were assembled automatically from 9 to 12 images taken at Â100 magnification with a fluorescence microscope (Olympus BX53). Afterward, the area covered with blood and lymphatic vessels was detected with an algorithm established in the image analyzing program Cell ^F (Olympus Soft Imaging Solutions GmbH, Münster, Germany), as previously described. 42 Briefly, before analysis, gray value images of the whole mount images were modified by several filters, and vessels were then detected by threshold setting, including the bright vessels and excluding the dark background. The mean vascularized area of control corneas was defined as 100%, and vascularized areas were then related to this value.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!