Peptide antibodies against three Sap-r-specific epitopes (designated Sap-rI, Sap-rII and Sap-r1, compare Fig. 1 A) were ordered from Pineda Antibody Service, Berlin, Germany. The obtained sera were affinity purified via Protein A-coupled Sepharose and tested on tissue and lysates (control versus mutant) and on Sap-r-overexpressing tissues. Secondary antibodies coupled to Alexa 488, Alexa 543 or Alexa 647 were from Molecular Probes and used at 1:200-1:1000. Filipin staining was done as described elsewhere (Huang et al., 2007 (link)). Fly tissue was dissected on ice and fixed in 4% formaldehyde in 1×PBS for 20-40 min or stained with LysoTracker Red/MitoTracker Green FM (both Molecular Probes) before fixation. After MitoTracker Green/LysoTracker Red double staining, as well as single LysoTracker Red staining, live tissue was mounted and imaged immediately. Imaging was done with a Zeiss LSM 710 microscope and images processed using Image J/Fiji and Photoshop software.
Alexa 543
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa 543 is a fluorescent dye used for labeling and detection of biological molecules in laboratory applications. It has an excitation maximum at 543 nm and an emission maximum at 568 nm, making it suitable for use with common fluorescence detection instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using alexa 543
1
Peptide Antibodies for Sap-r Epitopes
2
Immunohistochemical Analysis of mTOR and NeuN in Rat Brain Ischemia
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Immunohistochemistry was performed on sections of rat brain 24 h after ischemia as described.36 (link), 38 (link), 49 (link) Briefly, rats were killed under deep anesthesia by transcardial perfusion with 4% paraformaldehyde in PBS. Brains were immersion fixed in 4% paraformaldehyde in PBS overnight, washed with 30% sucrose in phosphate buffer solution, and frozen with 2-methylbutane. Frozen brains were cryostat sectioned at 20 μm. Sections were blocked and incubated for 48 h at 4 °C with antibody to mTOR and for 1 h with antibody to NeuN, followed by incubation with goat secondary antibodies Alexa 488 and Alexa 543 (1:500; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The specificity of immunolabeling was confirmed by incubation of sections with preimmune rabbit IgG in place of primary antibody; under these conditions, no detectable labeling was observed. Sections were viewed and images were acquired through a Zeiss DUO V2 laser scanning confocal fluorescence microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA). Images were processed with ImageJ software (Public Domain, imagej.nih.gov/ij/).
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