0–3 hour old embryos were collected from F1 adults from maternal RNAi crosses, dechorionated with 4.2% sodium hypochlorite for 2 minutes, then fixed in 4% formaldehyde and heptane for 20 minutes, and devitellinized by the addition of methanol and vigorous shaking. Fixed embryos were rehydrated by washing 4 times with PBSTx (PBS, 0.1% Triton X-100) and blocked with 10% bovine serum albumin (BSA) in PBSTx. Embryos were incubated with guinea pig anti-SMG (1:2000 dilution, 1% BSA in PBSTx) rocking overnight at 4°C and subsequently washed 3 × 15 minutes while rocking at room temperature. Embryos were then incubated in Cy3-conjugated donkey anti-guinea pig secondary antibody (1:300 dilution, 1% BSA in PBSTx; Jackson ImmunoResearch) for 1 hour rocking at room temperature, and washed 5 × 10 minutes with PBSTx. 0.001 mg/mL DAPI (Sigma) was added to the second wash to label DNA. Embryos were mounted in 2.5% DABCO (Sigma), 70% glycerol in PBS. Images were collected using a Zeiss AxioSkop-2 MOT fluorescence microscope and the QCapture Suite PLUS acquisition software.
Cy3 conjugated donkey anti guinea pig secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
The Cy3-conjugated donkey anti-guinea pig secondary antibody is a laboratory reagent used for detection and visualization in immunoassays. It consists of a donkey-derived secondary antibody that is conjugated to the Cy3 fluorescent dye, which emits light in the red-orange region of the visible spectrum when excited.
Automatically generated - may contain errors
Lab products found in correlation
Qcapture suite plus acquisition software, by Zeiss (2 mentions)
Axioskop 2 mot fluorescence microscope, by Zeiss (2 mentions)
Dabco, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Guinea pig anti rbpms, by PhosphoSolutions (1 mentions)
Alexa fluor 488 conjugated rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
To pro 3 iodide, by Thermo Fisher Scientific (1 mentions)
4 protocols using cy3 conjugated donkey anti guinea pig secondary antibody
1
Immunostaining of Embryonic SMG Protein
2
Quantifying Pancreatic Insulin Cells
3
Quantification and Visualization of Retinal Ganglion Cells
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To quantify and visualize RGCs, the whole-mount retinas were incubated for 6 days at 4 °C with primary antibody against the mouse RNA-binding protein with multiple splicing (RBPMS; 1:1000 dilution, guinea pig anti-RBPMS, PhosphoSolutions, Aurora, CO, USA), which is uniquely expressed in RGCs [19 (link)]. This was followed by incubation with 1:400 Alexa Fluor® 488 conjugated rabbit anti-GFP (Molecular Probes, Eugene, OR, USA) and Cy3 conjugated donkey anti-guinea pig secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) overnight at 4 °C. After that, the retina was rinsed in PBS for 10 min, then incubated in the nuclear counterstain TO-PRO-3 iodide (Thermo Fisher Scientific, Waltham, MA) for 15 min. Retinas were flattened with RGCs facing up, mounted with anti-fade fluorescent mounting medium (Sigma-Aldrich, St. Louis, MO), and coverslipped. Images were taken using a × 20 objective with a confocal microscope (Nikon C1, Nikon Canada Inc., Toronto, ON). Three images with an area of 330.32 × 330.32 μm from each retina were used for RGC quantification: near the optic disk, near the periphery and at an intermediate distance. Image J was used to perform RGC counts.
4
Immunostaining of Embryonic SMG Protein
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0–3 hour old embryos were collected from F1 adults from maternal RNAi crosses, dechorionated with 4.2% sodium hypochlorite for 2 minutes, then fixed in 4% formaldehyde and heptane for 20 minutes, and devitellinized by the addition of methanol and vigorous shaking. Fixed embryos were rehydrated by washing 4 times with PBSTx (PBS, 0.1% Triton X-100) and blocked with 10% bovine serum albumin (BSA) in PBSTx. Embryos were incubated with guinea pig anti-SMG (1:2000 dilution, 1% BSA in PBSTx) rocking overnight at 4°C and subsequently washed 3 × 15 minutes while rocking at room temperature. Embryos were then incubated in Cy3-conjugated donkey anti-guinea pig secondary antibody (1:300 dilution, 1% BSA in PBSTx; Jackson ImmunoResearch) for 1 hour rocking at room temperature, and washed 5 × 10 minutes with PBSTx. 0.001 mg/mL DAPI (Sigma) was added to the second wash to label DNA. Embryos were mounted in 2.5% DABCO (Sigma), 70% glycerol in PBS. Images were collected using a Zeiss AxioSkop-2 MOT fluorescence microscope and the QCapture Suite PLUS acquisition software.
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