About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon LAP2a siRNA knockdown (bottom row). Right panels: merged images with DAPI cellular stain. Credit: Courtesy of Vanja Pekovic" />About the image: The nuclear protein LAP2alpha (left panels) expresses a cell proliferation marker (K167, middle panels) when measured with a positive RNAi control (top row) but not upon

About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to a marked decrease in AP-3Δ levels. Credit: Courtesy of Raphael Rozenfeld" />About the image: Neuro2A cells transfected with a control siRNA (A) or a siRNA against the protein AP-3Δ (B), and stained 48 hours later with an AP-3Δ antibody (green). The transfection leads to

Related Articles Fine control: How to make sure your siRNA ducks are in a row Triple up and rescue Negative controls Order in the house mRNA versus protein Titrate siRNAs. Using the lowest possible concentration of siRNA may help avoid unwanted side effects, says Taylor. "Off-target effects can be dose-dependent." Before you start your study, run experiments with different siRNA concentrations to determine the level of knockdown you need to see a change in phenotype, Behlke say

With siRNA experiments, you need the right combination of controls before you can trust your results. Here's how to make sure your siRNA ducks are in a row.

About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor" />About the image: Biotin-labeled siRNA (brown) in mouse lung tissue. Credit: Courtesy of Catherine Taylor User: Catherine Taylor, University of Waterloo, Canada Project: Using siRNA knockdown to identify proteins involved in apoptosis Problem: As in Ruiz-Vela's case above, SiRNAs can induce off-target effects. Controls: Tayl

About the image: A colored scanning electron micrograph of a normal human myeloid leucocyte cell (right) and a leucocyte cell undergoing apoptosis (left). Credit: © Gopal Murti / Photo Researchers, Inc." />About the image: A colored scanning electron micrograph of a normal human myeloid leucocyte cell (right) and a leucocyte cell undergoing apoptosis (left). Credit: © Gopal Murti / Photo Researchers, Inc. User: Antonio Ruiz-Vela, Spanish National Cancer Center, Mad

How to make the most of multicolor immunofluorescence

Related Articles Chasing Rainbows How to Maximize Immunofluorescence Multiplexing Cutting crossover Unsticking Staying bright User: Pok Man Mendy Chan, Mount Sinai School of Medicine, New York The project: Studying expression of G-protein coupled receptors in striatal neurons and their involvement Parkinson's disease. The problem: Metabolically active tissue from postmortem brains in Chan's studies shows high levels of background fluorescence. The s

A mesenchymal cell labeled with three dye-conjugated antibodies and a nuclear stain. The top left image is a composite of the four markers. Credit: Courtesy of Matthias Schieker, University of Münich" />A mesenchymal cell labeled with three dye-conjugated antibodies and a nuclear stain. The top left image is a composite of the four markers. Credit: Courtesy of Matthias Schieker, University of Münich User: Matthias Schieker, University of Munich, Germany The p

Related Articles Chasing Rainbows How to Maximize Immunofluorescence Multiplexing Background glow Cutting crossover Staying bright User: Xiaohu Gao, University of Washington, Seattle The project: Characterizing tumor tissue by analyzing as many as five coexpressed proteins in clinical tissue biopsies. The problem: Gao wanted to use quantum dots (Invitrogen) because they allow easier multiplexing, but dots pose two serious aggregation problems. First, each qu

Related Articles Chasing Rainbows Background glow Cutting crossover Unsticking Staying bright Combine dots and dyes to label the most proteins simultaneously. By doing this, Mario Roederer's group at the National Institute of Allergy and Infectious Diseases in Bethesda, Md., can image 17 colors in its flow cytometry experiments. Stuart Sealfon of Mount Sinai School of Medicine recommends saving quantum dots for the proteins that are the most difficult to pick up, while using org

A confocal section of a cell labeled with three quantum dot-conjugated antibodies. Credit: Courtesy of Diane Lidke, University of New Mexico School of Medicine, Albuquerque" />A confocal section of a cell labeled with three quantum dot-conjugated antibodies. Credit: Courtesy of Diane Lidke, University of New Mexico School of Medicine, Albuquerque User: Diane Lidke, University of New Mexico School of Medicine, Albuquerque The project: Labeling as many as six proteins si