4. Return cells to incubator for 30 min. After the incubation
period, wash cell extensively (at least 3Â) with pre-warmed
media. The cells are now ready to be imaged using confocal
microscopy.
3.3 Bio-distribution
In Vivo
This protocol details how to perform bio-distribution studies for a
candidate CPP. Here we provide an example of bio-distribution
studies using Cy5.5-CTP in a mouse model. A number of organs
should be harvested, such as the heart, lung, liver, kidney, brain,
spleen, stomach, large intestine, small intestine, skeletal muscle,
bone, and testes/ovaries, with each time point performed in
triplicate.
1. Weigh mice and anesthetize with Ketamine/Xylazine (2 μL/g
of tissue weight) administered intramuscularly or intraperitoneally. Adequate level of anesthesia will be achieved in 5–7 min,
as assessed by lack of response to toe pinch.
2. Calculate a 10 mg/kg dose of Cy5.5-CTP, dilute to no more
than 200 μL, and inject either retro-orbitally or through tail
vein injection using an insulin syringe (see Note 2).
3. Allow peptide to circulate for the pre-specified time.
4. Euthanize mouse using Institutional Animal Care and Use
Committee’s specified method and open the chest cavity.
5. Place a nick in the right atrium and, using a 26G needle, inject
3 mL of 10% Buffered Formalin Phosphate for the dual purpose of perfusion fixing the organs of the mouse and flushing
out red blood cells.
6. Dissect out the organs of interest and store each organ individually in 10% Buffered Formalin Phosphate in a volume at least
20 times the volume of the tissue, weight per volume, for a
minimum of 48 hrs, light protected, at room temperature (see
Note 4).
7. Transfer organs into Tissue-Tek Processing/Embedding Cassettes and process the organs using a Tissue-Tek VIP processing machine.
8. Dehydrate the tissue in 70% EtOH for 30 min, followed by
80% EtOH for 30 min, 95% EtOH for 30 min, 95% EtOH for
30 min, 100% EtOH for 15 min, 100% EtOH for 20 min, and
finally 100% EtOH for 20 min.
9. Clear the tissue in xylene twice, 30 min for each xylene
treatment.
10. Infiltrate the cleared tissue with paraffin wax four times at
60
C for 30 min with each treatment.
11. Embed in paraffin using metal molds. Fill the mold with molten paraffin kept at 65
C and transfer to a cold plate. As the
paraffin at the bottom of the mold begins to solidify, place the
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period, wash cell extensively (at least 3Â) with pre-warmed
media. The cells are now ready to be imaged using confocal
microscopy.
3.3 Bio-distribution
In Vivo
This protocol details how to perform bio-distribution studies for a
candidate CPP. Here we provide an example of bio-distribution
studies using Cy5.5-CTP in a mouse model. A number of organs
should be harvested, such as the heart, lung, liver, kidney, brain,
spleen, stomach, large intestine, small intestine, skeletal muscle,
bone, and testes/ovaries, with each time point performed in
triplicate.
1. Weigh mice and anesthetize with Ketamine/Xylazine (2 μL/g
of tissue weight) administered intramuscularly or intraperitoneally. Adequate level of anesthesia will be achieved in 5–7 min,
as assessed by lack of response to toe pinch.
2. Calculate a 10 mg/kg dose of Cy5.5-CTP, dilute to no more
than 200 μL, and inject either retro-orbitally or through tail
vein injection using an insulin syringe (see Note 2).
3. Allow peptide to circulate for the pre-specified time.
4. Euthanize mouse using Institutional Animal Care and Use
Committee’s specified method and open the chest cavity.
5. Place a nick in the right atrium and, using a 26G needle, inject
3 mL of 10% Buffered Formalin Phosphate for the dual purpose of perfusion fixing the organs of the mouse and flushing
out red blood cells.
6. Dissect out the organs of interest and store each organ individually in 10% Buffered Formalin Phosphate in a volume at least
20 times the volume of the tissue, weight per volume, for a
minimum of 48 hrs, light protected, at room temperature (see
Note 4).
7. Transfer organs into Tissue-Tek Processing/Embedding Cassettes and process the organs using a Tissue-Tek VIP processing machine.
8. Dehydrate the tissue in 70% EtOH for 30 min, followed by
80% EtOH for 30 min, 95% EtOH for 30 min, 95% EtOH for
30 min, 100% EtOH for 15 min, 100% EtOH for 20 min, and
finally 100% EtOH for 20 min.
9. Clear the tissue in xylene twice, 30 min for each xylene
treatment.
10. Infiltrate the cleared tissue with paraffin wax four times at
60
C for 30 min with each treatment.
11. Embed in paraffin using metal molds. Fill the mold with molten paraffin kept at 65
C and transfer to a cold plate. As the
paraffin at the bottom of the mold begins to solidify, place the
106
Kyle S. Feldman et al.