1. Preparation and fabrication of tissue paraffin blocks
The tissue used to make the paraffin block should be fresh, the tissue fixative should be 10% neutral formalin, and the volume of the fixative should be more than 10 times the volume of the tissue. Insufficient tissue fixation, only superficial fixation, internal tissue decay over time, affecting results.
Formalin-fixed tissue cannot be stored for a long time. It is best to complete the embedding of paraffin blocks as soon as possible within 72 hours to ensure that the expression results of tissue proteins are not affected by experimental bias. If the next step of paraffin block embedding cannot be completed within a few days, it is best to store it in a refrigerator at 4 degrees, rather than at room temperature. Embedding in paraffin blocks requires dehydration and paraffin immersion. The quality of dehydration affects the fusion of tissue and wax in the later stage.
In addition, the principle of paraffin raw materials is also crucial. Faced with different tissues, it is necessary to dynamically adjust the paraffin composition. For parenchymal tissues with high toughness, high melting point paraffin blocks can be used, while soft tissues such as intestinal walls and glands can be used. Choose a paraffin block with a lower melting point. In addition, whether the base of the embedded paraffin block is flat or not directly affects the loss when the specimen is sectioned. Therefore, here is a reminder: the same paraffin block tissue may be due to the pit at the bottom of the paraffin block, resulting in a large amount of tissue loss during the trimming process.
2. Common ways to make paraffin blocks
(If it is a big brother research group, please skip this paragraph directly.)
Advantages: almost no extra cost, ready to use.
Disadvantages: Most of the small and medium-sized laboratories or research groups in China do not have the corresponding paraffin block production equipment.
2. Outsourcing options for paraffin block making
At present, most of the projects required paraffin blocks are outsourced. There are two main outsourcing objects: specialized experimental technology companies and service support related to basic medical pathology in universities. How to choose?
Specialized companies make paraffin blocks. The first is fast, and after all, it is driven by interests; second, the service is good, one phone call can be like sending express, without leaving home, pick up the goods and deliver them to the door; the third has the service industry. Advantages, except for those that may threaten direct economic interests, other supporting services, such as providing consultation and supporting materials, will be free. After all, domestic specialized experimental technology companies are still in the stage of market development, and there is no heavy bureaucracy or monopoly situation.
However, the services related to basic medical and pathology in universities are generally limited to the school, with limited productivity. Most of the time, they need to queue up, and the quality cannot be completely said to be high-quality. However, what is most topical, at the current reform stage, is the public services or platforms within these schools, which are constantly reformed to adapt to market and off-campus needs. However, generally speaking, the reform of price and charges is the fastest, which is in line with the market companies quickly, while the service quality will always lag behind, resulting in the status quo of “doing things difficult and expensive”. Therefore, if you want to lead your own paraffin blocks with peace of mind and dignity, it is best to support market-driven experimental technology companies. It is worth noting that experimental technology companies are different in the production of paraffin blocks, the use of raw materials and the compliance of protocols, so the quality of paraffin blocks is also uneven. The price ranges from RMB 10/piece to RMB 50/piece. In general, the number of encapsulation is huge, and it is easy to have problems after embedding. However, the company is the most cost-effective and most time-saving and cost-effective choice for sending a dozen to dozens of tissues for embedding paraffin blocks required for general topics.
3, the preservation of paraffin blocks
Unlike tissue frozen at -80 degrees, paraffin block tissue can be permanently stored at room temperature as long as it is sealed away from light. Sections obtained from paraffin blocks can be permanently preserved after HE or immunohistochemical staining, and have unique advantages compared to frozen sections and immunofluorescence.
4, how to maximize the utilization of small paraffin blocks
At present, the largest stock of paraffin block-embedded tissue is concentrated in the pathology departments of various hospitals, and these paraffin tissues are quite targeted clinical-molecular biology research materials. In addition to the above techniques, molecular biology techniques such as in situ PCR in paraffin sections are also important supplements to immunohistochemistry.
In today’s era of high-throughput genetic analysis, clinical specimens are often used in liquid nitrogen. With the advancement of molecular biotechnology, the use of paraffin-embedded tissue can also keep pace with the times, and can be used for genetic analysis, such as next-generation sequencing. Of course, the paraffin block used for genetic analysis also has a “shelf life”, and the best time for the paraffin block used is the fresh paraffin block within 1-1.5 years. If the paraffin block has been made for more than 5 years, the integrity of DNA and other information in the tissue will be greatly reduced, which will affect the processing accuracy and analysis process of later data.
In addition, by extracting tissue with the diameter of a pencil lead from several or dozens of paraffin block tissues and making a small tissue chip, it can not only simplify the time and cost of post-immunohistochemistry, but also save tissue to the greatest extent, and can also standardize Histochemical results. In general, after the promotion of chip technology, paraffin block tissue can be dynamically and selectively studied in batches, and gradually formed the “movable type printing technique” of tissue morphology.
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