organ in the desired orientation. Place a labeled cassette on top
of the mold as a backing and overfill with molten paraffin.
Allow to cool until completely solid.
12. The block can then be stored in a -20
C freezer over night to
completely solidify.
13. Prepare a 38
C water bath with distilled water.
14. Set up the microtome with a blade angle of 6
and a section
thickness of 15 μm.
15. Section the tissue in the microtome by cutting the desired
plane and then placing the blocks face down in the water
back for 5 min or until the tissue has absorbed some moisture.
16. Place the tissue onto a flat ice block for 10 min.
17. Place a fresh blade onto the microtome and cut sections with a
thickness of 8 μm. Discard bad paraffin ribbons until a ribbon
of sufficient length and quality is produced.
18. Quality ribbons are then picked up with forceps and floated on
the surface of the 38
C water bath. Let the sections sit on the
surface until they smooth out, taking care to not leave them
too long to prevent the paraffin from disintegrating and tearing
apart the section.
19. Float the flattened sections onto the surface of clean glass
slides.
20. Place the slides into a 65
C oven for 30 min to melt the wax.
These slides can be stored at room temperature with light
protection.
21. Deparaffinize the slides in Xylene three times, 10 min for each
treatment.
22. Rehydrate the tissue in 100% EtOH for 5 min, followed by 95%
EtOH for 5 min, 70% EtOH for 5 min, 50% EtOH for 5 min,
and finally 1Â TBS for 5 min.
23. Mount the slides with coverslips using 125 μL of Dapi
Fluoromount G.
24. Dry slides overnight at room temperature, light protected.
25. Image slides using confocal microscopy.
3.4 Ligand-Receptor
Coupling Experimental
Protocol for TriCEPS
This section provides details on how to perform an LRC-TriCEPS
experiment and contains protocols that can be modified for use in a
variety of cell lines and ligands of interest.
3.4.1 Ligand Coupling
The following protocol (Fig. 1) describes step-be-step an LRC
experiment using TriCEPS v.3.0 (azide-containing) for generating
one ligand and one control sample; therefore, the quantities need
to be multiplied by the number of desired biological replicates
(minimum three recommended) (see Notes 14–18).
Validating Cardiac Targeting Peptide
107
of the mold as a backing and overfill with molten paraffin.
Allow to cool until completely solid.
12. The block can then be stored in a -20
C freezer over night to
completely solidify.
13. Prepare a 38
C water bath with distilled water.
14. Set up the microtome with a blade angle of 6
and a section
thickness of 15 μm.
15. Section the tissue in the microtome by cutting the desired
plane and then placing the blocks face down in the water
back for 5 min or until the tissue has absorbed some moisture.
16. Place the tissue onto a flat ice block for 10 min.
17. Place a fresh blade onto the microtome and cut sections with a
thickness of 8 μm. Discard bad paraffin ribbons until a ribbon
of sufficient length and quality is produced.
18. Quality ribbons are then picked up with forceps and floated on
the surface of the 38
C water bath. Let the sections sit on the
surface until they smooth out, taking care to not leave them
too long to prevent the paraffin from disintegrating and tearing
apart the section.
19. Float the flattened sections onto the surface of clean glass
slides.
20. Place the slides into a 65
C oven for 30 min to melt the wax.
These slides can be stored at room temperature with light
protection.
21. Deparaffinize the slides in Xylene three times, 10 min for each
treatment.
22. Rehydrate the tissue in 100% EtOH for 5 min, followed by 95%
EtOH for 5 min, 70% EtOH for 5 min, 50% EtOH for 5 min,
and finally 1Â TBS for 5 min.
23. Mount the slides with coverslips using 125 μL of Dapi
Fluoromount G.
24. Dry slides overnight at room temperature, light protected.
25. Image slides using confocal microscopy.
3.4 Ligand-Receptor
Coupling Experimental
Protocol for TriCEPS
This section provides details on how to perform an LRC-TriCEPS
experiment and contains protocols that can be modified for use in a
variety of cell lines and ligands of interest.
3.4.1 Ligand Coupling
The following protocol (Fig. 1) describes step-be-step an LRC
experiment using TriCEPS v.3.0 (azide-containing) for generating
one ligand and one control sample; therefore, the quantities need
to be multiplied by the number of desired biological replicates
(minimum three recommended) (see Notes 14–18).
Validating Cardiac Targeting Peptide
107