This protocol provides a simple method of fixative perfusion in mice that will allow the histologic study of CNS tissues without significant investment. The main advantage of the gravity perfusion method presented here is that the profusion apparatus can be constructed with easily available components. Demonstrating the procedure will be Arnav Rana, an MD-PhD student from Dr.Xin Jie Chen's laboratory.
To begin, using a sharp razor blade, trim the inner straws from two 500-milliliter wash bottles by cutting them flush to the inner side of the screw on the cap. Cut a four-by-four-centimeter square hole in the bottom of each buffer bottle, then cut another small hole in the bottom of each bottle to allow the passage of a bent stainless steel micro spatula. Next, create an S-shaped bend in the micros spatulas so that they may be used as hooks for hanging the buffer bottles.
Insert the bent micro spatulas into the appropriate openings in the buffer bottles. Next, tightly connect two 25-centimeter-long tubes with the outer bottle straw outlets and join the ends of these tubes together with the Y connector, then join the 2-meter-long tubing to the free end of the Y connector. After removing the plunger from a 1-milliliter syringe, cut the syringe approximately 6 centimeters away from the tip by first scoring the plastic with a razor blade and then sharply snapping the syringe plastic.
Insert the cut face of the syringe into the free end of the plastic tubing to a sufficient depth and ensure a tight seal. Next, label one buffer bottle as PFA and the other as PBS. Measure approximately 1/3 of the length away from the bottle opening and draw a line around the bottle circumference at this point to denote the appropriate filling level of the perfusate solutions.
After straightening a large paperclip, create a loop that can fit around the circumference of the tubing and place this loop around the tubing, just proximal to the syringe, then place an appropriately-sized Styrofoam block in the glass tray, place the ends of the paperclip into the Styrofoam block to affix the tubing and using a paper towel, raise the front end of the glass tray by approximately 2 centimeters. After rinsing a 1-liter beaker with distilled water, fill it with approximately 800 milliliters of 18 milliohms molecular-biology-grade water. Heat the beaker in a microwave for three minutes or until the water temperature reaches 65 degrees Celsius, then place it on a heating or a stirring plate kept in a fume hood.
Next, rinse a stirring rod with distilled water and place it in the beaker. Start the stirrer and turn the hot plate up to medium heat, ensuring that the water temperature does not go above 70 degrees Celsius. After wearing a surgical mask, gloves, and lab coat, measure 40 grams of PFA powder and add it into the heated water, then using a transfer pipette, add a few drops of 5-molar sodium hydroxide to the solution.
Allow the powder to dissolve fully. If the powder has not fully dissolved, add more sodium hydroxide as needed. Once all the PFA has nearly dissolved, stop the stirring and heating and immediately add 100 milliliters of 10x PBS, then use the 18 milliohms molecular-biology-grade water to top up the beaker to the 1-liter mark.
Cover the beaker with plastic wrap and place it in a minus 20-degrees Celsius freezer until the solution reaches room temperature. After calibrating a pH meter using appropriate standards, measure the pH of the solution while the beaker is on a stirring plate. Add hydrochloric acid until the pH reaches 7.4.
If the pH is too low, add 5-molar sodium hydroxide to increase the pH. Next, connect a vacuum flask to vacuum and place a clean ceramic Buchner funnel with filter paper in the flask. After turning the vacuum on, wet the filter paper using a transfer pipette filled with the 4%PFA solution, then slowly pour the solution onto the filter paper until all the solution has been filtered.
After filtering, store the solution in a light-protected container at 4 degrees Celsius until use. In the fume hood, place a Styrofoam dissecting block in a glass tray. Ensure that the dissecting block has five to six short needles to restrain the mouse during surgery and two long needles to support the perfusion tubing.
Next, rinse the perfusion apparatus with distilled water. After all the water has drained, hang the profusion bottles one meter above the animal to be perfused, then prepare non-sterile 1x PBS using 10x PBS diluted with molecular-biology-grade-water. Clamp the main line of the perfusion apparatus using a hemostat, then clamp the PFA line with another hemostat.
After filling the buffer container with PBS, place the syringe end of the perfusion apparatus in a beaker to collect waste buffer and remove the hemostat occluding the main line. While the PBS flows through the line, vigorously tap on the walls of the tube to remove trapped air. Once all the air has been removed from the buffer and main lines, occlude the flow by placing a hemostat on the main line.
Next, remove the hemostat from the PFA line, allowing the PBS to flow in a retrograde manner up to the PFA bottle while tapping out any bubbles in the PFA line. Continue to allow the PBS into the PFA line until it can be seen just above the opening of the bottle, then occlude the PFA line with a hemostat to stop the flow of PBS into the PFA bottle. Next, connect the butterfly infusion needle to the perfusion syringe and open the main-line hemostat to flush PBS through the line and remove bubbles from the perfusion syringe.
After the bubbles have been removed, close the main-line hemostat. Ensure that the PBS bottle is now approximately 1/3 full of PBS. If necessary, flush PBS through the main line or fill more PBS into the buffer bottle until 1/3 of its full capacity.
Once all bubbles have been removed from the PBS, PFA, and main lines, fill the PFA bottle with 4%PFA solution at room temperature up to the black mark on the bottle. Next, prepare for non-survival surgery by cleaning the necessary instruments first with water and then with 70%ethanol. After securing an anesthetized C57 black 6J mouse to the Styrofoam block, begin the surgery by lifting the abdominal skin with skin forceps and cutting through the abdominal wall with large scissors.
Continue the abdominal cut superiorly towards the liver, then detach the liver from the anterior abdominal wall and continue the initial incision superiorly towards the diaphragm. When the incision reaches the diaphragm, using fine-dissecting scissors, make a hole in the diaphragm and cut through the ribs on the mouse's right side approximately halfway to the right axilla, then make a similar, but longer incision through the left side of the diaphragm almost all the way to the left axilla. Reflect the chest wall and pin it to the Styrofoam block.
After identifying the left ventricle, using fine-curved forceps, hold the heart steady with light pressure, then open the main-line hemostat to allow the PBS to flow through the needle and immediately pierce the left ventricle with the needle. Ensure that the needle is inserted no more than 0.5 centimeters into the ventricle. Next, rest the butterfly tubing on an X made in the Styrofoam with two large 18-gauge needles.
Identify the inferior vena cava as it exits the liver and transect it using fine-dissecting scissors. Continue PBS perfusion until the liquid flowing out of the inferior vena cava is blood-free, then working quickly, occlude the PBS line with a hemostat and open the PFA line. Allow the PFA to perfuse for a few minutes until the tail starts to curl.
Once the tail starts to curl, begin a 50-minute timer. After 50 minutes, occlude the main line with a hemostat and withdraw the needle from the left ventricle. Next, place the mouse in a labeled container of PFA solution at 4 degrees Celsius overnight.
High-quality perfusion is indicated by the absence of blood in the liver, spinal cord and deep central nervous system structures. The blood observed in the brain is located between the skull and the dura mater and is therefore not problematic or suggestive of poor-quality perfusion. Detection of alpha-synuclein following this perfusion method shows that phosphorylated alpha-synuclein accumulates significantly higher in 15.5-month-old symptomatic mice relative to seven-month-old asymptomatic mice expressing human A53T alpha-synuclein.
This result is consistent with reports describing an enrichment of cytopathology in the anterior horns of the spinal cord and the midbrain of these mice. It is important that no abdominal organs are cut when accessing the thoracic cavity. Furthermore, the perfusion needle must not be placed too deeply within the left ventricle.
