All the samples were obtained in accordance with the Declaration of
Helsinki with the Patients’ Informed Consent and with the Institutional
Review Boards’ approval. The cardiac tissue samples were obtained from
the patients undergoing heart transplants. The cardiac tissues were processed on
different ways depending on their further applications, as described, thus only
briefly outlined here [24 (link)–25 (link),51 ]: (1) as native myofibrils to retain their
antigenicity; (2) dissociated in single molecules for native electrophoresis and
blotting; (3) retained in the Wisconsin solution for primary cultures or
gradually frozen; (4) rapidly cryoimmobilized for retaining structural live like
architecture and antigenicity on cryosections; (5) snap frozen, crushed,
homogenized, and lyophilized to be used for cultures or denaturing
electrophoresis.
The myofibrils were prepared on the ways, which assured retention of the
native antigenicity. Strips of cardiac muscle tissue were brought to a stretched
or contracted state and clamped with the U shaped vascular surgery forceps. They
were immersed in the buffer solution (75mM KCI,10mM Tris pH6.8, 2mM EGTA, 2mM
MgCl2, 0.1mM PMSF, 0.1%TritonX-100). Thereafter, the tissues were
homogenized in a Polytron (Brinkman Instruments Co., Westbury, NY, USA) and a
Teflon glass homogenizer. The myofibrils were collected by centrifugation at
1,000 g for 5 min. The pellets were washed by cycles of suspension and
centrifugation. Finally, they were infused with the fresh buffer containing
50% glycerol and frozen at −20°C for storage. They were
thawed and rinsed with the fresh buffer before use.
Small cubes of the fresh cardiac tissues were disintegrated with the
sterile, surgical scalpel and plated onto the Petri dishes with the bottoms
covered by matrigel or cardiac tissue sections and filled with the DMEM
supplemented with serum and antibiotics. The primary cultures were grown in the
incubators maintaining 37°C, 10% CO2, and saturated
humidity. For storage, the tissue cultures were infused with DMSO or glycerol
and frozen gradually to retain their viability.
Alternatively, the cardiac tissue was inserted into the gold
planchettes. They were rapidly cryoimmobilized in the HPM 010 (Balzers,
Lichtenstein, EU). The frozen muscles were either sectioned in the frozen
hydrated state or cryo-substituted, infused with 2.3M sucrose, refrozen, and
sectioned on the cryoultramicrotome (Leica, Vienna, A, EU).
Alternatively, the rapidly cryoimmobilized samples were crushed and
homogenized. They were resuspended in the buffers and frozen or lyophilized to
the powders.
All these approaches assured preservation of native state of the cardiac
muscle protein antigenicity and architecture. All specimens were examined by
MPFS, IB, EELS, and EDXS [52 (link)].
Helsinki with the Patients’ Informed Consent and with the Institutional
Review Boards’ approval. The cardiac tissue samples were obtained from
the patients undergoing heart transplants. The cardiac tissues were processed on
different ways depending on their further applications, as described, thus only
briefly outlined here [24 (link)–25 (link),51 ]: (1) as native myofibrils to retain their
antigenicity; (2) dissociated in single molecules for native electrophoresis and
blotting; (3) retained in the Wisconsin solution for primary cultures or
gradually frozen; (4) rapidly cryoimmobilized for retaining structural live like
architecture and antigenicity on cryosections; (5) snap frozen, crushed,
homogenized, and lyophilized to be used for cultures or denaturing
electrophoresis.
The myofibrils were prepared on the ways, which assured retention of the
native antigenicity. Strips of cardiac muscle tissue were brought to a stretched
or contracted state and clamped with the U shaped vascular surgery forceps. They
were immersed in the buffer solution (75mM KCI,10mM Tris pH6.8, 2mM EGTA, 2mM
MgCl2, 0.1mM PMSF, 0.1%TritonX-100). Thereafter, the tissues were
homogenized in a Polytron (Brinkman Instruments Co., Westbury, NY, USA) and a
Teflon glass homogenizer. The myofibrils were collected by centrifugation at
1,000 g for 5 min. The pellets were washed by cycles of suspension and
centrifugation. Finally, they were infused with the fresh buffer containing
50% glycerol and frozen at −20°C for storage. They were
thawed and rinsed with the fresh buffer before use.
Small cubes of the fresh cardiac tissues were disintegrated with the
sterile, surgical scalpel and plated onto the Petri dishes with the bottoms
covered by matrigel or cardiac tissue sections and filled with the DMEM
supplemented with serum and antibiotics. The primary cultures were grown in the
incubators maintaining 37°C, 10% CO2, and saturated
humidity. For storage, the tissue cultures were infused with DMSO or glycerol
and frozen gradually to retain their viability.
Alternatively, the cardiac tissue was inserted into the gold
planchettes. They were rapidly cryoimmobilized in the HPM 010 (Balzers,
Lichtenstein, EU). The frozen muscles were either sectioned in the frozen
hydrated state or cryo-substituted, infused with 2.3M sucrose, refrozen, and
sectioned on the cryoultramicrotome (Leica, Vienna, A, EU).
Alternatively, the rapidly cryoimmobilized samples were crushed and
homogenized. They were resuspended in the buffers and frozen or lyophilized to
the powders.
All these approaches assured preservation of native state of the cardiac
muscle protein antigenicity and architecture. All specimens were examined by
MPFS, IB, EELS, and EDXS [52 (link)].
