In clinical and scientific research, the common type of frozen tissue section is to slice adipose tissue. In clinical, tissues with fatty content in the periphery mainly are lymph nodes, small breast cancer lumps, etc.; in scientific research, those are some skin tissues such as abdominal skin, neck skin, etc.
Adipose Tissue Section Using RWD Cryostat
(1) Tissue Pre-treatment
Usually, there is insufficient time in clinical practice to adequately process fatty tissues. The simple operation is to select the material, place the tissue on absorbent paper, press it gently, squeeze out the fat droplets as much as possible, embed it with the embedding agent, and then place them in the freezing chamber. In terms of scientific research, it’s different. In general, we have more time to remove the surrounding fat tissue and improve the quality of the sections.
(2) Pre-cool the Specimen Disc
After comparing the pre-cooling effects of placing the specimen disc at room temperature and in a freezing chamber, we recommend the first step is to pre-cool the dry one in the cool chamber. If the embedded tissue is at room temperature first and then put on the quick-freezing table, it will cause frost crystallization because of the big difference in temperature between the front and rear of the disc itself.
(3) Tissue Material Selection
A thickness of 2-3mm is recommended. Small thickness may make it difficult to select the material and also lead to the incomplete ones. On the other hand, if the thickness is excessive, it may be slow to be frozen. Of course, as long as the requirements are met, a thin material within 1-2cm would be desirable.
A larger sample disc facilitates to flatten the slides and improves freezing efficiency. The embedding material needs to be used properly. Firstly, excessive usage slows down the freezing. Secondly, curling may occur when the embedding material is pre-refrigerated, resulting in difficulties for subsequent slice flattened. If necessary, we can lightly press the heat extractor on the tissue surface to assist in freezing (note the timing of the light pressure, as applying it too early may cause sticking).
Before sectioning, it is essential to adjust the chamber temperature. Based on practice and feedback, cryostats with dual cooling systems (chamber cooling and specimen head cooling) usually generate better sectioning results than those with single-cabinet cooling systems. A low temperature is particularly required for tissue sections with high-fat content, which is even lower than the minimum temperature setting of the single-freezing-chamber cryostat. In such cases, we need to use alternative cooling methods to achieve the desired temperature. Liquid nitrogen cooling can be employed, but it is more expensive. Alternatively, cryostats with dual cooling systems can be utilized, where the temperature of the specimen head can be set within a range of -10℃ to -50℃. This ensures efficient sectioning of adipose tissue.
Minux® Cryostat with Dual Coolling System
We often observe that the staining of the tissue slice is not deep or cells degenerate under the microscope. There could be several reasons, but mainly due to the untimely fixation. Personal operation habits are partly responsible for the improper fixation.
To initially judge the quality of the sections, we habitually attach the slice and will wait for a while and then take a look at the effects.
This extremely short period causes tissue cell degeneration in dry air, especially in high-temperature environments, which has a more profound impact. It requires us to do slicing immediately and put the sections into fixative in time to fix.
What’s more, technical skills are required to be more professional, and the cryostat machine with stable refrigeration and temperature control function would be better.
There are many factors in achieving good adipose tissue sectioning but experience review is crucial. What we can do is to constantly summarize, learn from the practice and improve our sectioning techniques.