The overall goal of this procedure is to rapidly characterize the bio-distribution of fluorescently labeled compounds in whole animals. This method can be used to define the bio-distribution of protein based therapeutics and to determine the effects of targeting molecules on inspired distribution. The advantages of this technique include the generally low cost of the required reagents and the speed at which accurate comparisons can be drawn between different compounds.
Given then rapid progress in the development of biologics, these techniques provide a convenient and accurate way to determine the bio-distribution of compounds in various animal models. For ex vivo imaging of whole organs, first open the image acquisition software and click initialize. Under imaging mode, select the fluorescent check box.
Under exposure time, select auto. Under binning, select small and set the F/Stop to two. Select the appropriate excitation and emission filters for the labels used in the experiment.
Then select field of view B and set the target height to the appropriate depth according to the tissue being viewed. Once the temperature indicator turns green, place the tissues onto the black background mat, taking care that the samples are completely separated from one another. When all the tissues have been placed, transfer the mat into the imager with all of the samples within the selected area grid and click acquire to image the whole organs.
Then store the samples in PBS until further processing. Immediately after imaging, fill half of a 500 milliliter beaker with dry ice and add approximately 350 milliliters of isopropanol, so that there is sufficient liquid above the dry ice to partially submerge a freezing container. Next, fill a freezing container partially with OCT embedding medium, taking care that there are no bubbles.
Now remove the tissue of interest from PBS storage and dry the sample thoroughly, touching a lab wipe to the edges of delicate tissues to wick away the access liquid. Place the dried tissue gently into the OCT, taking care not to introduce bubbles under the tissue. Then add extra OCT until the sample is completely covered.
Now submerge the freezing container as in the isopropanol for 30 to 90 seconds until the OCT and the tissue are completely frozen and store the embedded tissue at minus 80 degrees Celsius for at least 12 to 16 hours. To prepare the slicing standards, first remove the ends of the barrels from 15, one milliliter, single use syringes, such that the entire length of the shaft is a single diameter. Next, add the protein used to treat the animals before the organ harvest to fresh OCT to make 15, two-fold serial dilutions, starting with the original protein concentration.
The protein used for the standards must be from the same labeled batch used to treat the animals. The protein labeling will vary in efficiency such that separate labeling of sample protein will not produce accurate standards. Invert the tubes to mix the OCT protein solutions.
Then allow the OCT to completely settle before inverting the tubes another 10 to 20 times. When a homogenous suspension has been obtained, draw an OCT standard into the first syringe, taking care to avoid bubbles and immediately dip the syringe directly into the isopropanol for 10 to 20 seconds, as just demonstrated. Allow the mixture to freeze completely within the syringe, then store the standard at minus 80 degrees celsius.
To acquire sections of the frozen organs, first place the tissue samples into the cryostat long enough for the samples to equilibrate to minus 20 degrees celsius. Then cut the tissues into 20 micron-thick slices, mounting the sections onto room temperature, glass slides as they are acquired. Once sliced, the samples will be easily effected by both light and temperature.
Therefore, after mounting, immediately store the sections in a light-proof slide box at negative 20 degrees celsius. To acquire sections of the standards, first warm an OCT standard syringe straight from minus 80 degrees celsius storage in a closed hand for three to five seconds, until the exterior of the shaft has warmed. Then depress the plunger until an approximately one centimeter piece of the embedding medium cylinder as exited the syringe.
Using a razor blade, remove the end of the cylinder and place the OCT standard perpendicularly onto the cryostat chuck, using fresh OCT to hold the standard in place. Finally, slice 20 micron-thick cylinder sections from the standard, collecting one sample from each dilution onto a single slide, so that one standard section represents the entire standard curve. Here, representative ex vivo images of whole organs from protein treated animals can be observed.
With the heat map overlays demonstrating the fluorescence levels in radiant efficiency within the tissues. By drawing regions of interest around the individual tissues and plotting those values on their corresponding standard curves, standardized fluorescence values can be generated for accurate comparisons of the relative concentrations of the different treatment compounds. For example, in this experiment, it is immediately clear that elastin like polypeptide or ELP delivery via intranasal administration, is the most effective combination for reaching the central nervous system.
To assess the bio-distribution of the compounds, sections can be acquired as just demonstrated for quantitative measurement of the compound within selected regions of the tissue of interest. From these data it is clear that while intranasal administration of ELP may result in high central nervous system concentrations, the distribution of the compound in this region is primarily in the olfactory bulbs and cerebellum. Conversely, ELP delivered intravenously demonstrates lower concentrations overall, but with an equal distribution throughout the brain.
Once proficient, this technique can be completed in two to three days if performed properly. While attempting this procedure, it's important to remember to properly handle the tissues and standards. Since it's development, this process has provided a way for researchers in the field of bio-therapeutics to explore targeted therapeutic delivery in whole animals quickly and effectively.
Following this procedure, other methods, like immuno-staining, can be performed to answer additional questions about the cell specificity and intracellular localization After watching this video, you should have a good understanding of how to characterize the bio-distribution and quantitative values of fluorescently labeled compounds.
