1 Animals, Diet, and Tissue Collection
a) Eight to ten weeks old apoliprotein E-deficient (ApoE−/−) mice (n = 4) are fed at libitum high fat diet to induce atherosclerosis development.
b) Following 12 weeks of high-fat diet, animals are exsanguinated under anesthesia by drawing total blood from abdominal aorta.
c) Mice hearts are harvested after inducing animal euthanasia by over-inhalation of isoflurane.
d) Unflushed hearts are immediately frozen in liquid nitrogen and kept at −80 °C until time of sectioning.
e) Tissue may also be embedded in mOCT if embedding must be done for small tissue to provide support for sectioning.
f) Prepare mOCT by adding 5 g of PVA 6-98 to 50 ml to make a 10% PVA 6-98 solution.
g) Solution is microwaved and then stir to dissolve the PVA 6-98 into solution.
h) Once the solution has cooled to room temperature add 4 mL of PPG 2000 and 50 mg of sodium azide (NaN3).
i) Cover and store at room temperature.
2 Tissue Preparation
a) A cryotome set at 10 μm thickness is used for tissue sectioning of the heart aortic valves at an optimal temperature between −20 °C and −27 °C.
b) Sections are positioned on ITO-coated glass slides with a fine bristle brush and kept at −80 °C until needed for IMS and/or additional analyses.
c) Slides with tissue sections are removed from −80 °C and dehydrated by immediately placing in a Nitrogen box with a continuous sweeping gas flow for 20 min to remove moisture.
d) A 2400 Dots per Inch (DPI) digital picture of the tissue section is captured using a flatbed scanner.
e) Hematoxylin & Eosin (H&E) staining may also be performed on a consecutive section of the same tissue to later superimpose against the IMS-derived color-coded image.
3 Matrix Application
a) A home-built sublimation chamber configured with a vacuum pump, cold trap, hot plate, and glass chamber is used to deposit matrix onto the tissue.
b) The tissue section is taped to the bottom of the chamber, to be suspended approx. 2 inches above a uniform layer of dry DHB matrix crystals.
c) Adjust vacuum pressure to 50 mTorr, while the hot plate is adjusted to reach a temperature of 150 °C.
d) Once pressure and temperature are stabilized, place the sublimation chamber onto the hot plate filled with sand to help distribute the heat transfer.
e) Deposit DHB matrix onto tissue section by means of sublimation.
f) Sublimation is allowed to take place for a total time of 7 min.
4 Analysis
a) An UltrafleXtreme MALDI TOF/TOF is used to acquire the molecular images.
b) The instrument is operated in reflector mode to generate positive ions covering a mass range of 300–1000 atomic mass units (amu).
c) Mix DHB matrix with peptide standard, caffeine, clozapine at a volume ratio of (9:1:1:1) respectively.
d) Hand spot 1 μL of mixture on a stainless-steel MALDI plate and allow to air dry.
e) Perform external mass calibration prior to initiating the imaging experiment.
f) The ion source accelerating voltage is set at 20 kV, ion source 2 voltage is 17.8 kV, lens voltage is 7.0 kV, and the delayed extraction time is 120 ns.
g) Operate the smartbeam II laser above threshold, at a repetition of 2000 Hz, accumulating 100 shots at every pixel.
h) Set the laser beam diameter to 10 μm and set pixel step size to 10 μm, in order to uniformly ablate the entire tissue surface.
i) Dilute ovine wool cholesterol and a phosphatidylcholine standards and co-mix with DHB matrix.
j) Hand spot 1 μL of standard on a stainless-steel MALDI plate and allow to air dry.
k) Select an intense pixel from the MS analysis.
l) Manual acquisition is performed utilizing an ion gate (+/−2 Da window) to isolate the target analytes from surrounding signals.
m) FlexImaging ver. 4.1 software is used to set up the analysis, as well as post process the results.
n) Activate normalization of spectra by using the Root Mean Square (RMS) algorithm.
o) Select a desired m/z from the averaged spectrum window to display single ion images.
p) To display multiple single ions by selecting those of interest.
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Reference
- Bhattacharya, S. K. (2017). Lipidomics. Methods in Molecular Biology, 1609.