Our protocol allows us to track bio-distribution and to confirm the tissue-specific internalization of a peptide of interest using two separate but complementary methodologies. We can use this protocol to maximize the data obtained from a single animal and to acquire quantitative axial fluorescence imaging data. Additionally, we can obtain qualitative microscope images.
After acquiring solid phase synthesized C-terminus amide cap CTPs with an N-terminus labeled with Cy5.5 from a peptide synthesis facility, make a one or 10 millimolar stock solution aliquot of CTP in dimethyl sulfoxide for storage at minus 80 degrees Celsius protected from light. On the day of the experiment, load a 10 milligram per kilogram dose of Cy5.5 CTP in 200 microliters of PBS into an insulin syringe. Weigh a mouse and intravenously deliver the peptide into the anesthetized six-week-old female CD1 mouse.
Allow the peptide to circulate for the pre-specified experimental time period before euthanizing the mouse using a high-flow CO2 chamber and using scissors to open the chest cavity and making a small nick in the lateral free wall of the right atrium. Use a five milliliter syringe equipped with a 26 gauge needle to perfuse three milliliters of 10%buffered formalin phosphate through the left ventricular apex to fix the tissues and to flush out any red blood cells. Then collect the heart, lung, liver, kidneys, spleen, large and small intestines, bladder, ovaries or testes, and brain into individual wells of a 12-well plate for ex vivo optical imaging.
For in vivo imaging of the CTP transduced images, start the image acquisition software and click Initialize to prepare the system for imaging. Open the imaging wizard and select Fluorescence and Filter Pair. Click Names, Dyes, Cyanide, and Cy5 to set the excitation and emission parameters.
And click Manual Settings to set the exposure to one second, the binning to small, the F/Stop to eight, and the field of view to 15 centimeters. Click Acquire and set the folder in which to save the samples. Add the appropriate experimental information in the edit image labels window and click OK.The acquired image will appear.
Set the units to radiant efficiency. Double-click the image and click Corrections and Adaptive Fluorescent Background Subtraction. Adjust the threshold to cover only the organs of interest with purple.
Alternatively, right-click and select Crop Area to draw a box containing all of the samples of interest. When all of the images have been acquired, transfer the samples into scintillation vials containing 10%buffered formalin phosphate in a volume of at least 20 times that of the tissue and store the tissues at room temperature protected from light for at least 48 hours. To quantify the images, set the units to radiant efficiency and the region of interest to four by three.
Adjust the region of interest box to evenly cover all of the wells and click Measure Regions of Interest. Then click Grid Region of Interest Measurements to match the cells of the region of interest and click Export to save the data in either a text or CSV file. When the organs are sufficiently fixed after a minimum of 48 hours, transfer the samples into tissue processing and embedding cassettes and load the cassettes into a tissue processing machine.
Set the processor to dehydrate the tissue with ethanol as indicated, followed by clearing with two 30-minute xylene treatments and paraffin infusion with four 30-minute paraffin treatments. After the last paraffin treatment, remove the tissues from the processing machine. Place the tissues in individual metal molds and line each mold with molten paraffin.
Place the molds onto a cold plate. As the paraffin at the bottom of the mold begins to solidify, place the organs into the paraffin. When all of the organs have been placed, place a labeled cassette on top of the mold as a backing and overfill the molds with molten paraffin.
Allow the paraffin to cool until solid before storing the blocks at minus 20 degrees Celsius overnight. The next morning, set up a microtome with a blade angle of six degrees and a section thickness of 10 micrometers and mount the tissue blocks into the microtome. Begin cutting until sections containing the tissue are obtained and place the blocks face down in a 38 degree Celsius distilled water bath for five minutes or until the tissue has absorbed some moisture.
When a thin white outline of the tissue appears in the block, place the tissue onto a flat ice block for 10 minutes before returning it to the microtome in the same orientation as it was just loaded. Begin obtaining slices allowing truncated sections to form long ribbons of six to 10 sections each. To ensure a successful tissue section acquisition, keep the block hydrated.
Return the block to the ice if the section quality is poor or begin to decline. Discard suboptimal paraffin ribbons until a high quality ribbon of sufficient length to cover a slide is produced. Use a blunt edge to transfer quality ribbons into a 38 degree Celsius water bath and let the sections sit on the surface of the distilled water until they just smooth out.
Float the flattened sections onto the surface of a clean glass slide and melt the wax in a 65 degree Celsius oven for 30 minutes. To deparaffinize the slides, treat the samples three times with xylene for 10 minutes per treatment. After the last xylene treatment, rehydrate the tissues with five-minute descending ethanol immersions as indicated, followed by a five-minute immersion in tris-buffered saline.
After allowing the slides to dry overnight, mount the sections with coverslips and 125 microliters of mounting medium supplemented with DAPI and dry the slides overnight protected from light before imaging by fluorescence microscopy. After fixation, the heart, lungs, liver, kidney, spleen, and brain from experimental and control animals are arranged in a 12-well plate for ex vivo optical fluorescent imaging. Upon conversion of the counts to radiant efficiency, the fluorescence data can be quantified for each set of organs.
It's important to note that different organs demonstrate auto-fluorescence in response to different excitation wavelengths. Shorter excitation wavelengths such as enhanced green fluorescent protein are associated with higher levels of auto-fluorescence especially in the liver and brain than far-red or near-infrared excitation wavelengths. Formalin fixation and paraffin embedding of tissue sections from each organ allows the immunofluorochemical analysis of cell markers of interest as well as the quantification of immune or stromal cells of interest within each tissue.
Adjust the imaging settings to avoid saturation and keep track of your settings, then apply the same acquisition settings to other samples in the same experiment for consistency and accuracy. Immunohistochemistry can also be performed on the tissue sections if a candidate cell-penetrating peptide co-localizes with a structure or protein of interest.
