The discoloration of wax leaf specimens is mainly caused by light, humidity, oxidation of plant components, and improper handling. The core avoidance strategy is to control environmental factors, optimize the production process, and do a good job in post production protection. The specific methods are as follows. 1、 Collection and preprocessing stageChoose an appropriate collection timeAvoid collecting under high light at noon (when plant cells have vigorous metabolism and are prone to rapid dehydration leading to pigment degradation), and prioritize collecting in the early morning or evening; After collection, immediately place it in a sealed fresh-keeping bag (temporary storage) to avoid prolonged exposure of plants to air and reduce the oxidation of pigments such as chlorophyll and anthocyanins.Targeted treatment of special plantsPlants containing polyphenols and tannins (such as tea leaves and acorn leaves): After collection, quickly boil them in boiling water for 10-20 seconds (to kill them), destroy the oxidase activity, and then suppress them;Succulent/succulent plants (such as Sedum and Portulaca): First, remove excess fleshy tissue, use absorbent paper to absorb surface moisture, and then press to avoid water retention and browning;Flower specimens: Gently separate petals and avoid overlapping when spreading to reduce discoloration caused by high local humidity. 2、 Compression drying stageIncrease the frequency of replacing absorbent paperChange the absorbent paper 2-3 times a day in a humid environment (at least once in a dry environment) to keep it dry and avoid mold growth or pigment hydrolysis of the specimen due to moisture retention; When changing paper, handle with care and do not rub the specimen to prevent cell damage and accelerated oxidation.Control and suppress the environmentThe suppression area should be ventilated, dry, and away from light (can be placed in a cool indoor corner), and avoid direct sunlight (ultraviolet rays can damage chlorophyll and cause yellowing of leaves); If the environment is humid, a desiccant (silica gel) can be placed next to the specimen holder to accelerate water evaporation.Moderate pressureThe pressure of the specimen clamp is moderate (it is advisable to fix the specimen flat without squeezing the organs). If it is too tight, it will cause cell rupture and pigment exudation; If it is too loose, the specimen will not have sufficient contact with the absorbent paper, and uneven drying will cause local discoloration. 3、 Disinfection and Binding StageChoose a gentle disinfection methodPrioritize low-temperature disinfection (freezing at -20 ℃ for 48 hours) to avoid damage to pigments caused by chemical fumigation (such as bromomethane); Ordinary teaching specimens can be exposed to short-term sunlight (1-2 hours), but it is necessary to avoid fading caused by prolonged exposure.Avoid using easily dyed materials for binding materialsUse neutral glue (such as white latex) or pure cotton needle and thread binding, avoid using acidic glue or colored string (acidic substances can corrode specimens, colored materials may fade and contaminate specimens); Select acid free cardboard for the table paper (to prevent acidic substances from seeping out after the paper ages, causing the specimen to turn yellow).

Immersion specimens have various applications in teaching:1、 Visual display of biological morphology and structurePlant teaching: In botany teaching, soaking specimens can effectively showcase the organs of plants. For example, when displaying immersed specimens of flowers, students can clearly see the number, shape, and color of petals, as well as the structure of stamens (including stamens and pistils). Soak specimens like lilies allow students to observe that they have 6 petals, 6 stamens, and 1 pistil, and can be stored for a long time, making it convenient for students to observe at any time without being limited by the flowering period. For the fruits and seeds of plants, soaking specimens can also display their external morphology and internal structure. For example, by soaking pea pod specimens, students can see the arrangement of pea seeds in the pods.Animal teaching: In animal science teaching, specimen immersion allows students to intuitively understand the appearance of animals. Taking fish as an example, immersion specimens can display the fish's body size, types of fins (dorsal fin, pectoral fin, pelvic fin, anal fin, and caudal fin), and their positions. Students can observe the morphological differences of fins in different fish species, such as the elongated dorsal fin of crucian carp and the wider dorsal fin of fighting fish. For some small invertebrates, such as earthworm specimens, their link structures can be clearly presented, helping students understand the characteristics of link animals.2、 Assist in explaining knowledge of biological classificationClassification display: Immersion specimens can serve as physical display materials for biological classification. For example, when explaining the classification of vertebrates, display immersion specimens from different classes (such as fish, amphibians, reptiles, birds, and mammals). By comparing the immersion specimens of fish (with gill respiration and fins) and amphibians (with gill respiration for juveniles, lung respiration and skin assisted respiration for adults, and limbs), students can more intuitively understand the main differences between different classes and master the basic knowledge of biological classification.Species diversity presentation: Teachers can use immersion specimens to display species diversity within a specific group. For example, when explaining the insect class, display immersion specimens of various insects such as locusts, butterflies, bees, etc. Students can observe differences in wing morphology (locusts have leathery forewings and membranous hindwings, butterflies have colorful and membranous wings) and mouthparts types (locusts have chewing mouthparts, butterflies have siphon mouthparts), and appreciate the richness and diversity of insect species.3、Help understand the ecological relationships of organismsFood chain display: A simple food chain model can be constructed for teaching through a series of immersion specimens. For example, by displaying immersion specimens of plankton, small fish, and large fish, teachers can explain the relationship between plankton as primary producers being preyed upon by small fish and small fish being preyed upon by large fish, allowing students to better understand the energy flow in the food chain and ecosystem.Biological habitat environment correlation: Immersion specimens can also be combined with an introduction to the biological habitat environment. For example, when displaying immersion specimens related to mangrove ecosystems, immersion specimens such as mangrove plants (such as okra), fiddler crabs, and mudskippers living in mangroves are also displayed, accompanied by pictures or videos of the muddy intertidal environment of mangroves, allowing students to understand the interrelationships between these organisms and their environment.

In the process of making biological sections, in order to avoid tissue autolysis or damage by bacteria, and preserve its physical structure, once the tissue block is taken out of the living body, it should be immediately and properly disposed of. The treatment is fixation, usually by soaking the tissue in chemicals to preserve its morphology and chemical characteristics as much as possible.  The chemicals used to immobilize tissues are called fixatives. So how are they classified?  According to the composition of the fixing solution, it can be divided into single fixation, double fixation and multiple fixation. First, single fixation is only fixed with glutaraldehyde or starvation tetroxide, generally used for single-cell samples or permeable samples. Second, double fixation refers to the first use of glutaraldehyde or paraformaldehyde for pre-fixation, and then use osmium tetroxide for post-fixation, this method has good effect and is widely used. Third, multiple fixation refers to the combined use of three kinds of fixatives to achieve the purpose of complementing each other. The main purpose of this process is to truly preserve every detail of the ultrastructure of the cell at the molecular level, so that the slice structure is clearer and the morphology is more complete. 

Many laboratory researchers are accustomed to storing tissue wax blocks for a long time at room temperature after sectioning, and the tissue after sectioning is prone to antigen loss for long-term storage at room temperature.  Some studies have found that when sections are stored at room temperature for more than 3 months, immunohistochemical staining results are weakened or even negative. Therefore, some scholars have carried out new and old slice preservation time control experiments. 11 different tissue wax blocks were selected and 10 pieces were cut continuously for a total of 110 pieces per case, and placed in room temperature air for 1 month, 3 months, 6 months, and 1 year, and then the control experiment was carried out with new sections. The experimental results showed that when stored at room temperature for 3 months after wax sectioning, the sensitivity of antigens to most antibodies decreased by 50%, and the antigen loss of some sections was more. Most antibodies did not show positive results after 6 months of sectional storage, and only a few sections had weak positive expression after storage for more than 1 year, especially nuclear expression antigen loss was more prominent.  The reason may be related to oxidation, so in practical work, wax sealing sections and -20 degrees cryopreservation tissue sections can be used to avoid antigen loss, or paraffin tissue blocks can be stored at 4 degrees, and then sectioned when used, which can greatly reduce the antigen loss rate.

Tissue microtome is a kind of equipment for pathological section analysis of human tissues, animal and plant tissues. Tissue microtome are generally divided into rotary microtome and frozen microtome. Rotary microtome: It is used to slice by rotating the hand wheel. The wax block table is embedded in the metal clamp seat that can move up and down in the groove, and cuts the flat slice by pushing forward with the micro screw. Some rotary microtome are equipped with three knobs and a fastening knob on the head, which can deflect and tighten it in all directions, so as to adjust the section of the wax block. The cutting angle of the slicer can be adjusted. Because this microtome uses a heavy and large slicer knife, it generally does not vibrate when cutting hard tissue. The slice thickness can be adjusted from 1-30 microns to any thickness by the knob, and each gradient is 1 or 2 microns. The advantage of the rotary microtome is that the body is heavy, so it is more stable than the rocking microtome. It is very suitable for cutting paraffin sections. It can be ideal for cutting continuous sections and also for cutting large tissue blocks. 

Frozen sectioning method is the most commonly used sectioning technique in histochemistry, especially enzyme histochemistry, fixed or unfixed tissue samples can be frozen section, fresh tissue frozen sections for tissue intracellular enzyme preservation is also a commonly used sectioning method in immunocytochemical research, can better preserve a variety of antigens, especially the antigenicity of surface antigens.  1. PreparationClean up the test bench and instrumentsTurn on the cryomicrotome and pre-reduce the cryomicrotome to the required temperature (-15~-20 °C)Prepare the processed slides, blades, glue and medicines  2. Clamp the frozen tissue block on the microtome holder, start the rough advance and withdrawal button, turn the knob, and smooth the tissue. 3. Adjust the thickness to be cut, according to different tissues, in principle, it is a cell-intensive thin cut, and the fiber multicellular thin can be slightly thick cut, generally between 5 ~ 10μm. 4. Adjust the anti-coil plate. The key to making frozen slices lies in the adjustment of the anti-roll plate, which requires the operator to be careful and accurately adjust it well and adjust it to the appropriate position. When slicing, cut out a complete, smooth slice. When the cut tissue adheres to a clean slide, gently apply a gentle force in one direction to avoid wrinkles during the tissue spreading process, ensure the integrity of the tissue structure and the beauty of the section.