Application of Cryostat as the Histology Equipment in Immunohistochemical Experiments
| November 22, 2022
Steps for the Preparation of Sections in Immunohistochemical Experiments
Histology equipment is essential in immunohistochemical (IHC) experiments. The 2 common types of sectioning methods employed in IHC experiments are paraffin sectioning and frozen sectioning. The steps of paraffin sectioning are: collection of a fresh specimen — dehydration (fixation, dehydration, clearing, wax infiltration) — paraffin embedding — slicing — staining, mounting — observation under the microscope; this process involves the use of basic histology equipment including the dehydrator, OCT compound, paraffin sectioning machine, staining machine, mounting machine, microscope and so on. The steps of frozen sectioning are: collection of a fresh specimen — frozen embedding — slicing — staining (can be done manually) — observation under the microscope. For this sectioning process, basic histology equipment includes flash freezer, frozen sectioning machine and microscope.
Common Problems of Immunohistochemical (IHC) Experiments
The requirements of IHC experiments depend on the types of specimens, antigens and sectioning methods. Meanwhile, the preparation of paraffin sections in IHC experiments involves complicated steps. The pre-treatment of tissues has high requirements as some antigens may be destroyed midway through the process, resulting in the step of antigen retrieval. The step of retrieval can be omitted in frozen sectioning as it does a better job in preserving the immunocompetence of antigens.
If you want to skip the step of antigens retrieval–in which case, frozen sectioning will become the method of choice. However, it will easily lead to the formation of ice crystals, inflicting a certain level of damage on the cell structures. Besides, slices prepared by frozen sectioning are thicker than those prepared by paraffin sectioning, leading to a direct impact on the section results. If the chamber of the cryostat doesn’t provide sufficient refrigeration, then a flash freezer will be used for supporting the frozen embedding process. At the same time, one has to consider how to accommodate the needs of junior laboratory technicians in order to ensure that everything will run smoothly at the start of the experiment. In this case, the cost of operating expenses for the laboratory could be pricey.
Advantages of using RWD Cryostat for cryosectioning in immunohistochemical (IHC) experiments
Solve the Problem of Thick Tissue Slices
1. The refrigeration output of the compressor supports temperature compensation twice at a time; PID temperature control limits the fluctuation of temperature in the chamber within a certain range and automatically restores the temperature to a normal level.
2. The premium cross roller bearings, five-phase high-resolution sampling motor, highly-dense spindle, injection slide render a highly-precise injection system.
3. Once the above criteria are satisfied, an ideal section result can be achieved without setting the section thickness to the maximum.
Reduce the Pressure Caused by Ice Crystal Formation
The formation of ice crystals is mainly attributed to the slow freezing rate in frozen embedding. Therefore, it is necessary for the equipment to be capable of coping with the requirement of flash freezing. RWD Cryostat provides a flash freezing stage which can reach -42℃ at its minimum, whereas the flash freezing point can even achieve -60℃ at its lowest temperature. Meanwhile, the chamber and specimen head carry out freezing separately, reducing the occurrence of ice crystals formation to ensure the quality of section results.
Low Cost of Operational Expenses
RWD Minux® FS800A possesses 2 semi-conducting flash freezing points and 15 regular freezing points to cater to the applications which involve huge volumes of tasks. Meanwhile, the effectiveness of the freezing stage is aligned with the international standard, producing a similar function akin to frozen embedding. In this case, the cost of frozen embedding will be much lower without the use of a flash freezer.
At the same time, it provides a log record for running an immediate and effective sectioning retrospective in order to optimize the sectioning criteria and enhance the quality of section results.
At the start of IHC experiments, some fresh laboratory technicians fail to satisfy the sectioning criteria due to unfamiliarity with the use of cryostat, and therefore undermine the quality of section results. Taking all these into consideration, it provides an automated slicing function and allows users to pre-set the sectioning criteria. With the automated slicing feature, the quality and consistency of the section results are guaranteed. In the meantime, the machine can be better adapted to newbies in the laboratory to ensure a highly- effective start of the experiments.