This protocol is the most efficient and cost-effective method for accurately labeling beta-amyloid plaques, as compared to amyloid-specific antibodies, which are currently the gold standard for labeling these plaques. This technique takes less time and is as equally effective as any of the commonly used amyloid labeling methods. Curcumin strongly binds to amyloid plaques and can be used as an imaging probe during PET scans of the brain and for relatively noninvasive screenings using retinal scanning.
Curcumin also binds to neurofibrillary or tau tangle in Alzheimer's disease, alpha synuclein in Parkinson's disease, and mutant Huntington's proteins in Huntington's disease. Demonstrating the procedure with Panchanan Maiti will be Zackary Bowers, a graduate student, and Alexandra Plemmons, a research scientist from my lab. After confirming a lack of response to pain reflex in an anesthetized 12 month old Alzheimer's disease model mouse, place the mouse in the supine position on a surgery tray and make an incision into the abdomen and through the diaphragm.
Insert a 21 gauge perfusion needle into the incision and make a small incision at the right auricle to allow the perfusion fluid to drain from the body. Use a gravity fed perfusion system to allow ice cold 0.1 molar PBS fluid to flow into the needle for five to six minutes at a 20 to 25 milliliters per minute flow rate. If the perfusion is performed correctly, a clear liver will be observed.
A proper animal perfusion is essential because if the blood is not removed completely, curcumin may bind to the blood vessels and result in a green fluorescent background signal. When all of the PBS has been delivered, switch the buffer valve to an ice cold 4%paraformaldehyde solution for eight to 10 minutes before using scissors and forceps to free the brain from the skull. Then use a spatula to place the brain into a vial of 4%paraformaldehyde at least 10 times the tissue volume for four degrees celsius storage until processing.
To obtain sections for cryostat sectioning, sequentially immerse the brain in graded sucrose solutions at four degrees celsius for 24 hours per concentration before using a cryostat at negative 22 degrees celsius to obtain 40 micrometer sections, placing 10 to 20 sections per well in a six well plate containing PBS supplemented with 0.02%azide as they are obtained. When all of the samples have been acquired, store the sections at four degrees celsius until further processing. To obtain sections for paraffin sectioning, dehydrate post-fixed perfused brain tissue with sequential graded alcohol solutions for two hours per concentration, followed by two one hour 100%alcohol immersions and two one hour xylene immersions at room temperature.
After the last xylene immersion, penetrate the tissue with a one to one xylene to paraffin solution two times for one hour per incubation at 56 degrees celsius in a glass conical glass covered with aluminum foil before immersing the tissues in 56 degree celsius paraffin for four to six hours. At the end of the incubation, use a rotary microtome at room temperature to obtain five micrometer thick sections placing the sections in a 45 degree celsius tissue water bath as they are collected. For curcumin labeling of amyloid beta plaques in cryostat tissue sections, rinse the sections with PBS three times for five minutes per wash before immersing the sections in 70%ethanol for two minutes at room temperature.
While the sections are incubating, dissolve one millimolar of stock curcumin in methanol and dilute the solution to a final working concentration of 10 micromolar with 70%ethanol. At the end of the incubation, immerse the sections in the working curcumin solution for 10 minutes at room temperature at 50 rotations per minute. At the end of the staining period, discard the curcumin solution and wash the sections with three two minute washes in 70%ethanol.
After the last wash, transfer the sections to polylysine coated glass slides and mount a cover slip onto each slide with an appropriate organic mounting medium. Then view the sections on a florescence microscope using a 10 or 20x objective and the appropriate excitation and emission filters. For histochemical labeling of deep paraffinized tissue sections, immerse the five micrometer thick sections in xylene two times for five minutes at room temperature per immersion, followed by rehydration with graded one minute alcohol solution immersions.
At the end of the incubation, wash the sections with graded alcohol solutions for two minutes per concentration followed by clearing with two five minute xylene immersions. After the second xylene immersion, mount each section on a microscope slide with a cover slip and an appropriate mounting medium for florescence microscopy imaging as demonstrated. For labeling of cryostat section specimens with anti-A-beta antibodies, wash the samples three times with fresh PBS per wash in individual wells of a 12 well plate before blocking any non-specific binding with 10%normal goat serum in PBS and 0.5%triton x-100 for one hour at room temperature.
At the end of the incubation, discard the blocking solution and incubate the samples with a-beta specific antibody overnight at four degree celsius and 150 rotations per minute. The next morning, wash the sections with three 10 minute washes in fresh PBS per wash, followed by incubation with an appropriate secondary antibody conjugated with a red fluorophore for one hour at room temperature protected from light. At the end of the incubation, wash the sections three times with PBS followed by one wash with 70%alcohol.
After the alcohol wash, incubate the sections with 10 micromolar curcumin for 10 minutes at room temperature followed by three one minute 70%alcohol washes. After the last wash, dehydrate the sections with 90%and 100%alcohol for one minute per concentration and clear the sections two times for five minutes per immersion with fresh xylene per incubation. Then mount and image the sections as demonstrated.
For intracellular amyloid beta co-localization, stain the sections with anti amyloid beta antibody followed by staining with curcumin at room temperature and 50 rotations per minute protected from light as demonstrated. At the end of the incubation, counterstain with an appropriate nuclear dye for 10 minutes at room temperature and 50 rotations per minute protected from light followed by three washes in PBS. Then mount and image the sections by fluorescence microscopy as demonstrated.
Although increasing the incubation time with curcumin slightly increases the fluorescence intensity of the A beta plaques, the number of observed plaques is not significantly different between one and five minutes of staining time. Curcumin can be used to stain A beta plaques and cryosectioned, paraffin embedded, and human Alzheimer's disease tissue samples. Labeling with A beta specific antibody before curcumin staining reveals that the curcumin is completely co-localized with A beta at the same plaques that bind the antibody.
After staining with curcumin labeled A beta oligomers, curcumin co-localization with the oligomers can be observed in A beta plaques. Similarly, curcumin also co-localizes with the A beta specific antibody in intracellular spaces, confirming that the stain can label intracellular A beta. Curcumin labeling is also more prominent than conventional amyloid binding dyes.
Curcumin derivatives found in tumeric extracts label A beta plaques comparatively to curcumin in human Alzheimer's disease brain tissue regardless of the type of mounting medium used. In addition, curcumin immunofluorescence signals and counterstaining intensity are maintained even after staining with typical nuclear stains and other cell markers. We met that constant sections should be thinner than 40 microns.
This training procedure should be kept under 30 minutes and the optimal curcumin concentration is one to two micromolar. Double leveling retarded biomarkers can be performed on the same tissue and can provide a greater specificity of which types of brain cells are being leveled. Given that curcumin can reduce the amyloid plaque load, the precise mechanisms of how this process occurs is currently being investigated at both the cellular and molecular levels.
