Abstract | Protocol
Although some methods are available for visualizing brain vasculature in rodents, few are applicable across species and disease models. This study describes a new method for the visualization of the vasculature lumen and endothelial cells and characterizes their morphology in normal and kainic acid (KA) treated rats. Herein, labeling was accomplished using Fluoro-Turquoise (FT), a novel reactive blue fluorochrome conjugated to gelatin. Strong blue fluorescence was observed throughout the brain vasculature following intra-cardiac perfusion with FT gel in normal animals. However, in the brains of KA treated rats (hippocampus, midline and ventral thalamus, piriform cortex), the vascular lumen was typically constricted, sclerotic and only faintly stained. FT gel can also be used to visualize endothelial cells in the mouse brain making it especially useful since there are currently no other markers available for visualizing these cells in mice. The advantages of FT gel over other markers can be attributed to its unique chemical and spectral properties. Specifically, FT is a very bright blue UV excitable dye that does not bleed through when visualized using other filters, making it ideal for multiple immunofluorescent labeling studies. Its brightness at low magnification also makes it ideal for low magnification whole brain imaging. Compared to alternative techniques for visualizing blood vessels such as India ink, fluorescent dye-conjugated dextran, the corrosion technique, endothelial cell markers and lectins, the present method results in excellent visualization of blood vessels.
Catalogue #: 1FT/2FT depending on size
Product Name: Fluoro-Turquoise
Manufacturer: Histo-Chem, Inc.
Chemical Identity: 4,4’-bis((1,3,5-triazin-2-chloro)amino)stilbene-2,2’-disulfonic acid, disodium salt
CAS #: None assigned
Molecular weight: 862
Appearance: yellow / tan colored powder
Fluoro-Turquoise is a reactive bright blue fluorochrome designed for the covalent labeling of certain proteins (e.g. gelatin) and polysaccharides (e.g. dextrans).
Although its spectrofluorometric profile has not yet been fully characterized, when used in conjunction with fluorescent microscopy, ultraviolet excitation results in a bright blue emission color. For Nikon epifluorescent microscopes, a UV-2B filter cube is typically used.
HAZARD IDENTIFICATION AND FIRST-AID MEASURES:
RTECS #: not assigned. To the best of our knowledge, no toxicity hazards are associated with this product. However, routine laboratory safety practices are recommended including wearing a mask when using in powder form and wearing gloves, when using in liquid form. In the event of contact with skin, wash immediately with soap and water. In the event of eye contact, immediately flush with copious amounts of water.
STOREAGE, USE AND DISPOSAL CONDITIONS:
It is recommended that Fluoro-Turquoise powder, as well as solutions of its conjugates, be stored in the refrigerator at around 5 degrees C. Spilled dye or its conjugates should be cleaned up with soap, water and paper towels while wearing gloves. Unwanted Fluoro-Turquoise should be poured down the drain and then flushed generously with water.
FIRE FIGHTING MEASURES:
Not flammable at atmospheric conditions.
Sumit Sarkar, James Raymick, Larry Schmued, Current Neurovascular Research, "In vivo demonstration of Fluoro-Turquoise conjugated gelatin for visualizing brain vasculature and endothelial cells and their characterization in normal and kainic acid exposed animals" (In Press)
Sumit Sarkar and Larry Schmued, Neurotoxicology, "In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains" 33 (2012) 436-443
Sumit Sarkar, Bryan Raymick, Larry Schmued, submitted to the Journal of Neuroscience Methods, "In vivo demonstration of Fluoro-Turquoise conjugated gelatin for visualizing brain vasculature and endothelial cells and their characterization in normal and kainic acid exposed animals" March 2013
SYNTHESIS OF FLUORO-TURQUOISE LABELED GELATIN:
- Dissolve 20 grams of gelatin (we use Sigma type A 300 bloom pig skin gelatin) in 1 liter of 0.9% NaCl solution at 60-65 degrees C.
- Dissolve 150 mg Fluoro-Turquoise into 20ml of 0.1M sodium carbonate vehicle.
- Add solution # 2 to solution #1 (above) in 1 ml increments while stirring and heating to 60 – 65 degrees C.
- Adjust pH to 9.0 by adding 2 M (9%) NaOH drop wise.
- Heat to 80 degrees C. for 10 minutes while maintaining the pH at 9.0.
- Allow to cool to 60 degrees C.
- Adjust pH to 7.5 by adding 10% HCl drop wise.
- Add distilled water to replace any lost by evaporation.
USE OF FLUORO-TURQUOISE CONJUGATED GELATIN FOR VASCULAR STUDIES:
- Exsanguinate animal with perfusion of heparinized 0.9% saline via the ascending aorta.
- Perfuse with 10% formalin (4% paraformaldehyde) in 0.1M neutral phosphate buffer.
- Flush with second 0.9% saline rinse.
- Perfuse with FT-gel solution. The temperature of the gelatin entering the aorta should be around 55 degrees C. In rodent studies, rats are typically perfused with a 200 ml volume of gelatin, while mice typically receive around 50 ml of the FT-gel solution.
- Remove brain and immerse in cold (about 5 degree C) 10 % formalin in 0.1 M phosphate buffer, plus 20 % sucrose (if brain is to be freeze sectioned).
- Allow to post fix and cryoprotect for at least 24 hr before sectioning.
- Mount tissue sections on gelatin coated slides from distilled water and air dry on a slide warmer at 60 degrees Centigrade for at least ½ hour.
- Clear slides by brief immersion in xylene prior to coverslipping with a non-polar mounting media such as DPX.
- Visualize bright blue vasculature labeling using an epifluorescent microscope with ultraviolet light excitation.
SYNTHESIS OF FLUORO-TURQUOISE LABELED DEXTRAN:
- Dissolve one gram of 10,000 MW dextran-amine (Invitrogen) in 10 ml distilled water.
- Dissolve 75 mg of Fluoro-Turquoise in 10 ml of 0.1 M sodium carbonate.
- Add solution #2, above, to solution # 1 while stirring.
- Adjust pH to 9.0 by adding 2M NaOH drop wise.
- Heat for one hour at 70 degrees C. Adjust to pH 9.0, if necessary.
- Allow to cool to room temperature.
- Add dye solution to 400 ml of absolute alcohol while stirring.
- Centrifuge at 3,900 RPM for 20 minutes.
- Discard supernatant, collect and dry precipitate on hot plate at 75 degrees C. overnight.
USE OF FLUORO-TURQUOISE LABELED DEXTRAN FOR THE IN VIVO LABELLING OF BRAIN VASCULAR PERICYTES FOLLOWING I.C.V INJECTION:
Fluoro-Turquoise dextran can be injected into the lateral ventricles to label all vascular pericytes in the brain. In this sense it can be used like another fluorescent dextran, Fluoro-Ruby (In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains, S. Sarkar and L. Schmued, Neurotoxicology, 33 (212) 436-443). The two main differences between Fluoro-Ruby and Fluoro-Turquoise are their respective emission colors (red vs. blue) and the fact that Fluoro-Ruby will undergo anterograde axonal transport, while the Fluoro-Turquoise will not.
- Using stereotaxic surgery, inject 1 ul of 10 % Fluoro-Turquoise dextran into both lateral ventricles just anterior to the confluence of the third and lateral ventricles.
- Allow animal to survive for 1-7 days.
- Perfuse anesthetized animal with saline followed by neutral buffered 10% formalin (4% paraformaldehyde).
- Remove brain and immersion post fix over night in same fixative solution (plus 20 % sucrose for brains destined to be freeze sectioned).
- Section brains and mount tissue sections on gelatin coated slides.
- Dry slides on slide warmer, clear by brief immersion in xylene and coverslip with non-aqueous, non-polar mounting medium, such as DPX.
- Examine slides under epifluorescent microscope using UV excitation to localize Fluoro-Turquoise dextran labeled brain vascular pericytes.