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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Perfusion of the animals
- Prepare the fixative and perfusion buffers.
- Prepare 0.1 M sodium phosphate buffer by adding 80 g of sodium chloride (NaCl), 2 g of potassium chloride (KCl), 21.7 g of disodium hydrogen phosphate (Na2HPO4·7H2O), 2.59 g of potassium dihydrogen phosphate (KH2PO4), and double distilled water to make a total of 1 L.
- Prepare 4% paraformaldehyde (PFA).
- Add 40 g of paraformaldehyde to 1 L of phosphate-buffered saline (PBS) (0.1 M, pH 7.4).
- Heat the PFA solution to 60−65 °C and mix using a magnetic stirrer.
NOTE: The temperature should not exceed 65 °C. - Add few drops of sodium hydroxide (NaOH) (1 N) with a dropper to dissolve the PFA completely.
- Filter the PFA solution with medium to fine filter paper and store at 4 °C.
NOTE: The solution is good for a month.
- Perform animal anesthesia and perfusion.
NOTE: Twelve-month-old B6SJL-Tg APP SwFlLon, PSEN1*M146L*L286V, 1136799Vas/J ( 5x familial Alzheimer's disease (5×FAD)) age-matched control mice (n = 6 per group) were purchased from vendors and bred in the animal house of Saginaw Valley State University. Genotyping was confirmed by polymerase chain reaction (PCR). Human Alzheimer's disease (AD) brain tissue includes postmortem AD brain tissue and age-matched control tissue.- Anesthetize the animal with an appropriate anesthetic agent, such as sodium pentobarbital (390 mg/kg body weight), or a ketamine/xylazine mixture (up to 80 mg/kg body weight ketamine and 10 mg/kg body weight xylazine) by intraperitoneal injection (27 G needle and 1 mL syringe). Check the level of anesthesia by pinching a toe. If the animal is unresponsive, then it is ready for perfusion surgery.
- Place the anesthetized animal in the supine position on the perfusion surgery tray and, using small iris scissors, make an incision to the posterior end of the left ventricle.
- Insert a 22-G perfusion needle into the left ventricle and make a small incision at the right auricle to remove perfusion fluid from the body. Use a gravity-fed perfusion system to allow the ice-cold perfusion fluid (0.1 M PBS, pH 7.4) to flow for 5−6 min (flow rate 20−25 ml/min).
NOTE: A clear liver is the indicator of optimum perfusion. - Switch the buffer valve to an ice-cold 4% paraformaldehyde solution for fixing and allow it to flow for 8−10 min.
NOTE: Tremor followed by hardened or stiff limbs is an indicator of good fixation. - Remove the brain from the skull using scissors. Using a spatula, collect the brain and place it in a vial of 4% PFA (at least 10x the volume of the brain) and store at 4 °C until further use.
2. Tissue processing
- Cut cryostat sections.
- Transfer the brain to graded sucrose solutions (10%, 20%, and 30%) and store at 4 °C for 24 h each, until use.
- Using a cryostat at -22 °C, cut 40 µm-thick sections. Collect 10−20 sections per well in a 6-well plate filled with PBS and sodium azide (0.02%).
3. Colocalize cur(curcumin) with the Aβ antibody in Aβ plaques and oligomers.
- Wash cryostat sections from step 2.1.2 with PBS 3x in a 12-well plate.
- Block the sections with 10% normal goat serum (NGS) dissolved in PBS with 0.5% Triton-X-100 at room temperature for 1 h.
- Discard the blocking solution. Incubate the sections with amyloid-beta (Aβ)-specific antibodies (6E10 or A11, diluted 1:200) dissolved in fresh blocking solution containing 10% NGS and 0.5% Triton-X100 overnight at 4 °C in a shaker at 150 rpm.
- Discard the antibody solution and wash the sections with PBS 3x for 10 min each.
- Incubate with the secondary antibody tag with red fluorophore (e.g., Alexa
- 594) for 1 h at room temperature in the dark.
- Wash with PBS 3x for 10 min each.
- Wash with 70% alcohol 1x.
- Incubate the sections with curcumin (Cur (10 µM)) for 5 min at room temperature.
- Wash with 70% alcohol 3x for 1 min each.
- Dehydrate with 90% and 100% alcohol for 1 min each, clear with xylene 2x for 5 min each, and mount on slides using distyrene plasticizer xylene (DPX).
- Visualize using a fluorescence microscope with appropriate excitation/emission filters for the red and green signals.