A cryoclamp for the rapid cryofixation of the isolated blood‐perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

Abstract

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non‐uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build‐up of sodium in this region could be an important factor in the development of ischaemia‐reperfusion injury. Electron probe X‐ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.

Immunocytochemical strategies for electron microscopy: choice or compromise

“…An adequate depth of freezing of 10 µm is all that can be expected from hand‐held cryo‐pliers. The automated mechanical devices were developed to improve the reproducibility of freezing and to capture time‐resolved events (Bond et al ., 1994; Shattock et al ., 1998). They may offer a small improvement in the depth of good freezing but they are unlikely to improve on the performance of the static impact freezing devices.…”