The overall goal of this sublimation protocol is to provide a detailed, easy-to-follow sample preparation process, for the detection of gangliosides in MALDI imaging mass spectrometry experiments. This method can help answer key questions in the neurobiology field, including the anatomical distribution and function of gangliosides, both in the healthy brain and in brain pathology. The main advantage of this technique is that it can be adapted for high-quality imaging of many lipid species few alterations to the protocol.
To begin this procedure, extract brain tissue from the rat and prepare to cryostat as described in the text protocol. Then position the tissue in the cryostat. Section the tissue up to the desired anatomical location.
Ensure that the cutting thickness is between eight and 12 microns. Using the cryostat anti-roll bar, slowly section flattened tissue section. Holes or markings on the tissue can occur if the roll bar placement is incorrect or if the blade is dull.
Move the tissue to the center of the slicing platform using clean paint brushes. To map the tissue, freeze conductive slides or metal plates by placing them in the cryostat while slicing. When the slide is completely frozen, carefully move the section tissue onto the conductive service of the slide using clean paint brushes.
Once all tissue section are positioned correctly on the slide, place a finger under the slide opposite the tissue and press until the section thaws. Then place the slides with tissue in a desiccator for five to 10 minutes. Alternatively, use a warm slide and lightly press it, conductive side down, against the flattened tissue sections.
Working in the fume hood, place a sand bath in an aluminum container onto a hotplate. The hotplate should be on a metal scissor lift of the appropriate surface area. Turn on the hotplate and set the temperature to 140 degrees Celsius.
Use the temperature feedback probe to monitor sand temperature throughout the experiment, and ensure temperature consistency. Next, place 300 milligrams of DAN matrix onto the bottom surface of the sublimation apparatus. Spread the matrix out in an even layer in the center of the apparatus to the approximate width and length of the slide being sublimated.
Place a metal plate onto the inner surface of the apparatus with the plate making direct contact with the bottom of the condenser. Tape a blank test slide diagonally cross the surface of the metal plate, with a tape placed on the outer edges of the slide. Connect the top and bottom portions of the apparatus with a rubber O-ring in the middle to ensure complete seal.
Place a metal U-joint around the center of the apparatus, and tighten vices until the top and bottom half of the apparatus are sealed tightly together. Then place the apparatus into the metal O-ring about the sand bath. Next, place a handful of crushed ice in the condenser.
Fill the condenser one-fourth to one-half full with cold water to create ice slush. The ice slush will cool the metal plate on the inside of the apparatus, and subsequently the slide adhered to it. Wait at least five minutes for the temperature to reach a steady state.
Then, pour 300 mL of methanol in the cold trap container and place the glassware into the container. Connect the vacuum pump to the cold trap output using rubber tubing. Use another piece of rubber tubing to connect the cold trap input to the sublimation apparatus.
Ensure that the tubing is tightly secured using metal clamps. Use the vacuum pump to deliver a vacuum of 30 to 50 millitorr. Allow the pumps to run for at least five minutes for pressure equilibration.
Drop two to three small pieces of dry ice into the ethanol, in the bottom of the cold trap container. The cold trap helps prevent matrix particles from accumulating in the vacuum pump. Vices on the U-ring of the sublimation apparatus may need tightening once the vacuum pump is turned on because of decreased pressure in the apparatus.
Ensure that the sand bath temperature has stabilized at 140 degrees Celsius. Also check that the vacuum is on and that the apparatus is secured in the metal O-ring above the sand bath with all tubing connected. Set the timer for seven minutes but do not start the timer.
Slowly raise the scissor lift and sand bath up to the sublimation apparatus until the U-joint of the sublimator is well above the metal O-ring. This extra space allows for adjustment of the apparatus on the sand. Quickly press the sublimator gently on the sand surface to ensure that the apparatus is sitting evenly on the sand.
And then, immediately start the timer. When the timer sounds, turn off the vacuum pump and carefully lower the scissor lift until the sublimator is no longer touching the sand bath and sitting securely in the metal O-ring. Slowly loosen the microvent valve to release pressure in the apparatus.
Loosen the metal clamp around the rubber tubing of the sublimation apparatus, and slowly begin to loosen the tube. Once the rubber tube has been loosened slightly, bend the tube to one side to allow residual pressure to escape. When ambient pressure returns, carefully remove the rubber tubing from the sublimation apparatus.
Next, loosen the vices on the U-joint of the apparatus and remove them. Carefully separate the two halves of the sublimation apparatus and pull-off the slide from the top of the inner glassware. Examine the slide to confirm even matrix distribution.
If the distribution and amount of matrix sublimated is sufficient, tape a new slide with tissue onto the inside of the sublimator and repeat the sublimation process. Proceed to perform imaging and analysis as described in the text protocol. The mass spectrum displays ionic abundance of anolytes within a 1000 to 2000 mass range.
The major A-series gangliosides are labeled along the spectrum. Shown here is the corresponding molecular image of gangliosides in the rat brain. The molecular image shows the anatomical distribution of several ganglioside species through color coding, which have been overlaid and pseudocolored using ImageJ software.
Generally, individuals new to this method may struggle, as there are many variables in the sample preparation process which can influence the quality of IMS results that are often left out of reported protocols. While attempting this procedure, it's important to remember to monitor the amount of matrix being sublimated after each experiment, and modify the time of sublimation accordingly for the slide. After its development, this technique paved the way for researchers to explore the role of membrane lipids such as gangliosides, after brain injury, ner-jone disease and the natural aging process.
Those this method can provide insight into the abundance and anatomical distribution of lipids in the brain, it can also be applied to other systems, including tissues outside the brain, and even plant and insect models. Working with damaged roots can be extremely hazardous and precaution such as gloves, masks and eye protection should always be taken when performing this procedure.
