Prepare the hexane/dry ice bath by half-filling a large rectangular bowl with hexane (size depends on sample; bowl must be large enough to contain hexane/dry ice solution and allow the sample to be fully submerged), then carefully adding dry ice pellets to the hexane (CARE, see Note 3 ). The amount of dry ice pellets required will vary depending on size of the bowl and ambient temperature, etc. Dry ice should be added until the solvent is cold and some pellets of dry ice remain floating in the hexane.

The euthanized rodent is placed in a frame to ensure alignment before freezing (Fig. 1a). If a frame is not available, gently pulling the head and tail in opposite directions to get the spinal column in line using forceps will achieve a similar outcome. Pinning to a cork board can also be used if required.

While maintaining the correct alignment submerge the rodent in the hexane/dry ice bath until completely covered. Fully freeze the sample; the time required varies depending on sample size. For instance, a typical male Sprague-Dawley rat (ca. 260 g) should be frozen for 30 min.

The freezing mold with stage (Fig. 1b, c) is precooled by placing on a bed of dry ice; a small amount of prechilled CMC is added to the bottom of the mold. The frozen carcass is then placed into the center of the container and the surrounding area is filled with prechilled (4 °C) 2% CMC (w/v). Ensure the container is at least three-quarters submerged and leave in the hexane/dry ice bath for 30 min or until fully frozen (the dry ice will evaporate over time and will need to be topped up as required). Once frozen the sample can be removed from the container and stored in a sealed bag at −80 °C until required.

Relative quantitation of exogenous compounds (e.g., pharmaceutical agents) can be achieved using blood or tissue homogenates spiked with the compound of interest at concentrations covering the expected range. This standard curve can be added to a CMC block by drilling holes in the block and filling these with spiked homogenates and freezing in the hexane dry ice bath. (N.B. at this stage the sample can be stored in a sealed bag at −80 °C until required).

Ensure the surface of the aluminum mounting plate is clean and flat (see Note 4 ).

Coat the mounting plates with the UV curable adhesive supplied with the Macro-Tape-Transfer-System (Instrumedics Inc., St. Louis, MO, USA) following the manufacturer’s instructions. Skip this step if using direct tape mounting without a tape transfer system.

For whole-body sectioning a specialized cryomacrotome is required (e.g., Leica CM3600 XP); smaller samples can be sectioned using a standard cryomicrotome.

Securely mount the block into the holder in the cryomacrotome, ensuring the correct orientation for sectioning (sagittal/coronal).

Using a fresh blade (care: the blade is extremely sharp) for each sample start to trim in the sectioning using large steps 50–100 μm until the required tissue depth is reached.

Set the cryomacrotome to 30 μm section thickness; cut and discard the next section.

Apply the adhesive tape to the top of the sample block and ensure it is firmly adhered using a rubber or foam hand roller.

Slowly cut the section while applying slight pressure a few millimeters in front of the cutting blade to prevent the tape lifting off the tissue. When cutting the section gently lift the tape with the attached section as it is being cut, ensuring not to pull the tape because the section can easily be damaged.

Cutting a second serial section for histological staining can be very useful for comparison with the MALDI image and will provide cellular information. (This section should be mounted to a glass slide for staining.)

Repeat steps 3–7 for all the tissue levels required.

If using direct tape mounting stick the sample tissue side up directly on the target plate using double-sided tape (skip steps 10–12).

Place the tissue section face down onto the precoated mounting medium, ensuring good adhesion using a hand roller.

Following the manufacturer’s instructions polymerize the adhesive using a pulse of long wave UV light .

Carefully remove the tape from the tissue section, leaving it firmly attached to the mounting medium.

Remove the section(s) from the cryostat chamber along with a metal block at least the size of your sample and place directly in the freeze-drier chamber. The metal block prevents the section(s) from thawing between the transfer and the start of the freeze drying process (the very thin tissue section would rapidly thaw if not kept on a frozen metal block).

Immediately evacuate the freeze dryer and dry the section for 45 min. (Fr4eeze-dried sections can be stored in an air-tight container or bag at −80 °C until required. However, when defrosting before use ensure the container/bag remains sealed until the sample has reached room temperature to prevent condensation forming on the tissue section).

Prepare the MALDI matrix solution (Subheading 2.5, item 2) and sonicate for 15 min before use (see Note 5 ).