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“Immunohistochemistry (IHC)” is a technique that uses staining reagents (such as fluorescein, enzyme, metal ions, and nucleotides) to label the specific antigens generated from the specific binding reaction between antigens and antibodies in order to determine the presence of antigens in cells of a tissue section for quantitative, qualitative and semi-quantitative analysis.
The application of immunohistochemistry is prevalent when it comes to pathological diagnosis, particularly tumor diagnosis and differential diagnosis. Generally speaking, IHC is necessary under two possible conditions. First, it is unable to identify whether the mass is cancerous or not, and therefore further clarification is needed. Second, the mass is already identified as cancerous, but the source and the type of cancer cells require further studies. Cryosectioning is preferred for IHC as it involves fewer steps and less equipment. Also, the antigenicity can be better preserved and antigen retrieval is no longer necessary.
Equipment
Cryostat is the essential equipment commonly used in IHC specifically due to its exclusive features, including strong refrigerating power and stable temperature control that render it the best working partner of IHC cryosection specialists.
Read more about Cryosectioning Protocols: Application of Cryostat as the Histology Equipment in Immunohistochemical Experiments
Process
(1) Extract the sample (tissue) and then place it onto a specimen disc. Embed the tissue with a small amount of OCT compound (or ultrasound coupling agent or glue), and then attach the tissue block to the cryostat chuck (snap freezing chuck can be opened in case of emergency or ample samples). Together with the stationary heat extractor, the sample is compressed to achieve the snap freezing effects.
(2) Place the specimen disc onto the specimen holder after the tissue is completely frozen. With the double cooling/refrigerating feature, the temperature of the specimen holder can be adjusted based on the types of tissues, delivering better sectioning results.
| Types of Tissues | Specimen Holder Temperature For Reference |
| Typical tissues, tumors, etc. | -19~-15℃ |
| Delicate tissues e.g. brain, liver and thyroid tissues | -15~-13℃ |
| Tissues with fats | -35~-25℃ or even lower |
(3) Roughly trim the sample to obtain a flat tissue surface;
(4) Start cutting the tissue with an anti-roll plate (or hold one end of the section with forceps and cut it while pulling). Generally, the section thickness should be maintained at 5~6um. After adhering the dissected tissue to the glass slide, carry out fixation immediately with an alcoholic fixative (such as methanol/acetic acid, 95% ethanol/acetic acid, pure methanol, 95% ethanol, etc.) for 10-20 seconds. Then, rinse it with water.
(5) Stain the tissue block with hematoxylin (room temperature) for 1-2 minutes; Then, rinse it with water.
(6) Differentiate the tissue block for 1-2 seconds by using 0.5%~1% hydrochloric acid ethanol; then, rinse it with water.
(7) Wash it with warm water to obtain the blue hue. (11) Stain it with eosin for 1-2 seconds.
(8) Carry out rapid dehydration through a graded series of ethanol. Clear it with xylene, and make it coverslipped with neutral resin.
Apart from the cryosectioning protocols above, there are important precautions for cryosectioning.
(1) Temperature of the Cryochamber
| Types of Cryostat | Features | Cryochamber Temperature For Reference |
| Single Cooling/Refrigeration Design | Only one chamber for cooling | Winter (low room temperature): around -18℃; Summer(high room temperature); around -21℃ |
| Double Cooling/ Refrigeration Design | Cryochamber and specimen holder undergo cooling separately | Winter (low room temperature): around -15℃ Summer (high room temperature): around -18℃ |
The temperature of the cryochamber is also subject to the nature of the tissues, the room temperature and the workload of sectioning. It is good to go as long as the tissue is not brittle and is easy to cut.
(2) Temperature of the Specimen Holder
| Types of Tissue | Specimen Holder Temperature For Reference |
| Typical tissues, tumors, etc. | -19~-15℃ |
| Delicate tissues e.g. brain, liver and thyroid tissues | -15~-13℃ |
| Tissues with fats | -35~-25℃ or even lower |
When the tissue is brittle, take the specimen holder out of the chamber and warm it for 3-4 seconds before putting it back for sectioning. Also, gloves can be worn to touch the tissue block. However, keep in mind that a small amount of ice crystals may form during the process due to changes in temperature, especially the formation of intranuclear ice crystals.
(3) Keeping the cryostat power on is the best way to avoid damages caused by frequent reboots.
OCT embedding compound is commonly used for embedding but the amount of OCT should be as minimal as possible because the smaller amount, the faster the freezing rate. As a result, there will be fewer ice crystals formed in the tissue. The hardness, shrinkage, tensility, and freezing rate of the embedding medium are all critical factors. Some embedding media may be more elastic which results in uneven staining. If the embedding medium causes severe shrinkage, the surface of the embedded tissue will not be smooth. Since the area of the tissue is always larger than the area encircled by the embedding medium, the embedded parts may form small blobs, and thus become wrinkled when being adhered to the slide.
Use disposable histological knives that are highly tensile, sharp and resistant to wear.
Snap freezing is the key to frozen sectioning, as only rapid freezing can reduce the number of ice crystals formed in the tissues. A slow freezing rate encourages water molecules to move towards intercellular regions (such as liver sinusoids, renal tubules, etc.) and accumulate, leading to the formation of ice crystals that causes the cells or tissues to expand. After quickly putting the section in the fixative, the ice crystals melt into water, causing the surrounding tissue to shrink. This creates a lot of holes in the tissue and leads to the deformation of tissue morphology. Therefore, only snap freezing can convert water molecules into ice before they leave the cell, keeping the cytoplasmic tangible matter in place.
Key points for snap freezing:
(1) Put the sample on the freezing stage for precooling
(2) The tissue sample should not be too large or too thick. Its thickness should be kept around 1mm with a size of 10-20mm. The larger and thicker the tissue, the slower the freezing rate.
(3) Use as little embedding agent as possible while ensuring that the tissue remains attached to the specimen disc, as a large amount of embedding agents can affect the freezing performance.
(4) Gently press the stationary heat extractor on the tissue till it separates from the extractor prior to sectioning.
(1) For tissues containing higher water content, such as brain tissue and soft tissue sarcomas, the cryochamber temperature should be set within -14~-13℃. If time allows, soak the tissue in a sucrose-saturated solution (high osmotic solution) for 24 hours. If time is limited, use the filter paper to remove water from the tissue. Brain tissues are prone to being brittle, and thus require more time to warm back up to the temperature around -14℃ (the operation will be more convenient if the specimen holder has a temperature control feature) before sectioning.
(2) Cartilage tissues (e.g. trachea cartilage), skin tissues, and tissues rich in glial and connective tissues are vulnerable to rolling or falling off the slide during staining. Thus, it is important to cut them thinner and use adhesive/positively-charged histological slides that can help reduce the occurrence of the above issues.
(3) Lymph nodes and small lumps of breast cancer tissue carry a large amount of fats. Place them between absorbent paper and gently press them with your fingers to squeeze out the fatty droplets before sectioning.
References
“A Concise Manual of Pathology Techniques” by Ding Wei, Wang Detian, Dong Jianqiang, Zhao Yiling, Wang Wenyong, and others – Zhejiang Science and Technology Press
A comprehensive cryostat guide provides in-depth information on cryostat machines, cryosectioning techniques, applications, precautions, and selecting the right machine. It aims to equip researchers and medical professionals with the knowledge and understanding necessary for successful cryostat applications in various fields.
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