1. 23W!~ Anti-vWF Immunofluorescence in Mouse Brain Minjung Choi, Katie Stockdale, James O. McNamara Department of Internal Medicine, The University of Iowa, Iowa City, IA. Abstract Flourescence tag and Filter properties How Flourescence Microscopes Work How Immunostaining Works ’ Conclusions Anti-VWF Labeling of Brain Section with AF594-Coupled Secondary Antibody Anti-VWF Labeling of Brain Section with AF633-Coupled Secondary Antibody AF 633 Filter GFP Filter AF 633 Excitation/Emission AF 633 Filter set AF 594 Excitation/Emission GFP Filter Excitation/Emission AF 594 Filter set GFP Filter set Brain Tissue vWF Primary Antibody Secondary Antibody Conjugated to Fluorophore Excitation Emission  What is immunostaining and how does it work? Antibody-based methods to detect a specific protein. In this case, primary antibody was designed to bind to the vWF and secondary antibody to the primary antibody. The secondary antibody is conjugated to a fluorescent molecule, which can yield specific signal. The signals were detected with a fluorescent microscope.  What is von Wilebrand Factor (vWF)? vWF is a large multimeric glycoprotein in blood plasma that is highly expressed on brain endothelium (inner cells of blood vessel).  How were brain sections prepared? The brain was obtained from Amyloid Precursor Protein (a protein implicated in Alzheimer’s disease) mutant mouse. Then the brain was perfused, fixed and cut into 10μm thick sections. The blood-brain barrier (BBB) blocks molecules larger than about 500 Daltons from escaping the brain vasculature and accessing the neurons and glia of the brain. While the BBB serves an important protective role for the brain, it also blocks access of most therapeutic reagents and is thus an obstacle for the treatment of many disorders of the nervous system. To determine whether intravenously administered experimental therapeutic reagents are able to cross the BBB in animal models of disease, it is necessary to determine the location of the reagents relative to the vasculature; methods for labeling the vasculature are useful for such studies. Here, we carried out immunofluorescent labeling of the vasculature in mouse brain tissue sections with an antibody specific for von Willebrand Factor, a protein expressed on the surface of brain endothelial cells. We explored fluorophores with red and “far red” emission spectra because brain tissue exhibits a lower level of autofluorescence at longer wavelengths. We obtained comparable results with secondary antibodies coupled to either red or far red emitting fluorophores. 1.Light source emits light with many different wavelengths. 2. Excitation Filter only allows a fraction of the wavelengths to illuminate specimen. 3. Dichroic mirror reflects the exciting light. 4. Fluorophore absorbs energy and emits light with longer wavelength. 5. Dichroic mirror only lets through the light with longer wavelengths. (The reflected, excitation light with short wavelength can’t pass through) 6. Emission Filter only allows the light within narrow range (this light has longer wavelengths than excitation light. 7. The filtered light, specific from target protein is detected by the microscope. Solid line: Emission Range of Flurophore(AF633) Dotted line: Excitation Range of Fluorophore(AF633) Solid line: Emission Range of Flurophore(AF594) Dotted line: Excitation Range of Fluorophore(AF594) Red: Excitation Filter range Green: Emission Filter range Blue: Dichroic range GFP Filter set was used to show that the signals can’t be detected with filter set with different filter range. Blue: Excitation Filter range Red: Emission Filter range Green: Dichroic wavelength Blue: Excitation Filter range Red: Emission Filter range Green: Dichroic range Primary antibody- anti von Willebrand Factor Secondary antibody- Alexa Fluor 633 No Secondary antibody Primary antibody- anti von Wilebrand Factor Secondary antibody- Alexa Fluor 594 No Secondary antibody AF 594 Filter GFP Filter Image is adapted from abcam.com http://en.wikipedia.org/wiki/Fluorescence_microscope An immunofluorescence protocol for labeling blood vessels of mouse brain tissue sections was developed. Anti-vWF labeling with secondary antibodies coupled to two different fluorophores (Alexa Fluor 594 and 633) was demonstrated; these fluorophore/filter combinations may prove useful in double-labeling experiments. The two fluorophore/filter combinations yielded comparable results. Lab Members