Àlog10 scale). Proteins that are enriched in one of the samples will
plot either left or right of the x-axis origin, indicating in which
sample the target protein is enriched.
4 Notes
1. H9C2 and 3T3 cells should be passaged once they are about
70% confluent. Do not allow to grow to complete confluency
as they will begin to differentiate.
2. Avoid freeze-thaw cycles of peptides once the lyophilized powder is in solution in DMSO. Aliquot into amber/black Eppendorf tubes and store long term (6–24 months) at À80
C.
Lyophilized powder can be stored longer term in À20
C, as
long as it is light protected, for fluorescently labeled CPPs.
3. Similarly, avoid freeze-thaw cycles of the Live/Dead stain once
reconstituted into DMSO.
4. Organs can be stored long term in formalin, or after being in
formalin for 48 h, can be transferred into 70% EtOH for longterm storage, with light protection for both.
5. Tissue processing machines can be programmed to perform the
series of solution exchanges needed to process tissue automatically though hand processing is a perfectly acceptable alternative provided the samples are protected from light.
6. Tissue blocks can be popped out of the molds and stored at
room temperature for years with light protection.
7. Do not oversoak tissue until it is swollen. Soaking until the
edges of the tissue lighten is sufficient.
8. When selecting fluorophores for tracking a CPP, care should be
taken to minimize spectra overlap, which can be an issue where
multiple fluorophores are needed. Another factor in selecting
fluorophores is the system being used in the experiment.
9. Minimize tissue autofluorescence and possible false-positive
results by choosing fluorophores in the red or far-red spectra.
10. Avoid the dye TAMRA, which can produce very high background due to membrane association.
11. When using multiple cell types in flow cytometry; differences in
cell size may need to be corrected for to ensure the results of
the experiment are valid. This can be addressed by dividing the
measured fluorescence by the measured forward scatter, a measure of cell size. Doing this would produce a measure of the
density of fluorescence per cell. This method could be used to
compare the results from multiple cell types.
110
Kyle S. Feldman et al.
plot either left or right of the x-axis origin, indicating in which
sample the target protein is enriched.
4 Notes
1. H9C2 and 3T3 cells should be passaged once they are about
70% confluent. Do not allow to grow to complete confluency
as they will begin to differentiate.
2. Avoid freeze-thaw cycles of peptides once the lyophilized powder is in solution in DMSO. Aliquot into amber/black Eppendorf tubes and store long term (6–24 months) at À80
C.
Lyophilized powder can be stored longer term in À20
C, as
long as it is light protected, for fluorescently labeled CPPs.
3. Similarly, avoid freeze-thaw cycles of the Live/Dead stain once
reconstituted into DMSO.
4. Organs can be stored long term in formalin, or after being in
formalin for 48 h, can be transferred into 70% EtOH for longterm storage, with light protection for both.
5. Tissue processing machines can be programmed to perform the
series of solution exchanges needed to process tissue automatically though hand processing is a perfectly acceptable alternative provided the samples are protected from light.
6. Tissue blocks can be popped out of the molds and stored at
room temperature for years with light protection.
7. Do not oversoak tissue until it is swollen. Soaking until the
edges of the tissue lighten is sufficient.
8. When selecting fluorophores for tracking a CPP, care should be
taken to minimize spectra overlap, which can be an issue where
multiple fluorophores are needed. Another factor in selecting
fluorophores is the system being used in the experiment.
9. Minimize tissue autofluorescence and possible false-positive
results by choosing fluorophores in the red or far-red spectra.
10. Avoid the dye TAMRA, which can produce very high background due to membrane association.
11. When using multiple cell types in flow cytometry; differences in
cell size may need to be corrected for to ensure the results of
the experiment are valid. This can be addressed by dividing the
measured fluorescence by the measured forward scatter, a measure of cell size. Doing this would produce a measure of the
density of fluorescence per cell. This method could be used to
compare the results from multiple cell types.
110
Kyle S. Feldman et al.