Plant paraffin sectioning technique
Plant paraffin sectioning technique
Due to their large size and opacity, plants cannot be viewed directly under a microscope. To study the internal structure of the plant, the thickness and volume of the sample must be reduced to allow light to pass through the sample so that it can be observed under a microscope. After processing the samples through various sectioning methods, the samples are not only small, thin, intact, transparent, and retaining their original structure, but also have color, which makes them easy to identify and observe. Paraffin sectioning is one of the most important and commonly used methods in microscopy. It is to use paraffin as embedding agent, and use rotary slicer to cut the samples into 8~12 pm thick continuous slices. After a series of treatments, permanent slices can be made.
The whole process is as follows:
Sampling and fixation, washing, dehydration, clearing, wax penetration, embedding of all slices, sticking, dewaxing, staining, dehydration, and clearing and fixing (Figure 8-1).
Operation method
Plant paraffin sectioning technique
Materials and Instruments
Equipment: blade (double-sided blade or razor), dropper, brush, petri dish, slides, goblet, warmer, slicer, brush, slides, baking table, staining vat. Move The basic process of plant paraffin sectioning technique can be divided into the following steps: -Sampling and fixation Select fresh, robust, normal and representative materials, divide them into small pieces or slices of 0.5~1 cm3 and quickly put them into the fixative for fixation. The purpose of fixation is to kill the tissues or cells quickly, so that their morphology and structure can be kept as close to the living state as possible. The most commonly used fixative is FAA or FPA [formaldehyde: propionic acid: 70% ethanol = 1:1:18, V/V]. For delicate samples with high water content, 50% ethanol can be used instead of 70% ethanol. The amount of fixative is usually 50 times that of the sample. When fixing, the air should be extracted from the sample to facilitate the entry of fixative into the sample. The fixation time is 2~24 h. It can also be stored in FAA fixative for a long time. Washing After fixation, wash away the fixative remaining in the tissue with water or ethanol. All fixatives prepared with aqueous solution should be washed with water. Any fixative prepared with ethanol is washed with the same concentration of ethanol. The washing time is generally 1~2 h, during which the detergent is replaced 2 or 3 times. Dewatering After the material is washed, the dehydration should be converted from low concentration of ethanol to high concentration of ethanol gradually to remove the water in the sample, the concentration of ethanol at all levels is 30%, 50%, 70%, 80%, 90%, 100% ethanol, 1~2 h for each level, the length of time depends on the size of the sample. If the wax cannot be buried temporarily, the material can be stored in 70% ethanol for a long time. Cleaning (Transparent) As the material is dehydrated, which contains ethanol, ethanol and paraffin wax can not be mixed, still need to be removed. De-ethanol usually use xylene, the material from ethanol into xylene, it is best to do it gradually, first by anhydrous ethanol xylene mixture, and then into the pure xylene, pure xylene need to be changed 1 or 2 times, each time 30 min. V. Wax penetration Take paraffin wax with a melting point of 52~56 ℃, cut it into small pieces with a scalpel, and put the wax pieces into a small wine cup with an equal amount of xylene. Then put the wine cup with the material into the 40 ℃ warm box, after 6~10 h, and then move into the 60 ℃ warm box for 1~2 h. During the process, the xylene gradually evaporated and the paraffin wax liquid became thicker, and then pour away the paraffin wax and pour in the melted pure paraffin wax, and then pour away the paraffin wax and replace it with the new paraffin wax after 2~4 h. After 2~4 h again, then the wax can be embedded. Embedding Prepare 1 pair of tweezers, 1 basin of cold water, alcohol lamp and lighter, and then according to the need to fold a few embedded carton, carton is folded into a hard and smooth paper, the size depends on the material. The first clean paraffin poured into the carton, with a hot set of paraffin wax in the air bubbles quickly driven off, and paraffin hot evenly, then pour the material, with a hot embellishments will be the material according to the required cut surface and a certain interval (both 3~4 mm) arranged neatly, to make the whole material to put flat (Figure 8-1). After the material is put in place, blow gently to make the surface of paraffin condense, and then put the carton flat into cold water (or on a cold plate) to make the paraffin solidify as soon as possible, otherwise it will make the paraffin crystallize, and the crystallized paraffin will not be able to be sliced. Made of wax stripped carton, labeled, can be stored for a long time. Slicing Split the wax block so that each small wax block has only 1 sample, then repair the small wax block into a positive hexahedron, when repairing the block to be the first part of the cut to leave a little wax, the opposite side of the wax to be more, and then stick the small wax block on the wood block, sticking when the small wax block is coated with a layer of melted wax first, and then stick the small wax block to leave more wax on the end of the wood block, and then melt a little bit of wax in the small wax around the block with a dissecting knife, so that the wax block is reinforced. It is reinforced. During this process, try to keep the cut side of the material perpendicular to the knife's edge. Clamp the block of wood with the wax block glued to the fixture of the rotary slicer, then mount the steel knife (or blade) on the slicer and adjust the angle of the block so that the cut surface of the material is parallel to the knife. Then adjust the thickness controller, dial to the required thickness, at this time the right hand shakes the slicer, after the wax block to the knife, the slices fall from the knife, due to the friction of the slicing process of heat, cut the slices connected to a wax band, at this time the left hand to take a brush will be the wax band to support, and sequentially put the wax pieces on the wax band disk. Sticking the slices The cut wax tape should be glued to the slide. Before gluing the slides, the wax tape must be covered to prevent dust from entering the slides; if the dust sticks to the slides, it will blur the structure of the slides made in the future. A small drop of paste is placed in the center of a clean slide, and this paste is spread evenly on the slide with the finger, followed by 1 drop of 8% aqueous formaldehyde or distilled water. Using a scalpel, cut the wax band to the desired size and pick it up and place it in the water droplets on the slide, placing it in such a way that the wax band has both smooth and rough sides, and sticking the smooth side down to the slide. Next, place the floating slide with wax tape on the baking table, and the wax tape will be unfolded by heat. During the process, move the wax tape to the position of 2/5 of the whole slide with an inlay, and then use absorbent paper to remove the excess water and allow it to dry, or put it in a warm box at 30~40 ℃ for 1 day to dry. Nine, dewaxing After drying the slides with slices attached, put them into xylene for 5~10 min in spring and fall. After the paraffin wax is melted away, the sliced material will still be adhered to the slides, and then the slides will be put into 1/2 anhydrous ethanol + 1/2 xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, and water in order. The above steps were carried out in a staining vat for about 1 min per stage. X. Fanshong and Solid Green Dyeing Method The red is prepared as a 1% solution in water, and the green is prepared as a 0.5% solution in ethanol (with 95% ethanol). After de-waxing in xylene, rehydrate from high concentration to low concentration of ethanol to distilled water, then stain with 1% saffron aqueous solution for 1~2 h. After washing in distilled water, dehydrate from 35%, 50%, 75% to 85% ethanol, and stain with solid green staining solution for 10~40 s, and then transition with 95% ethanol, anhydrous ethanol Ⅰ, and anhydrous ethanol Ⅱ in turn. Dewatering, transition through 1/2 anhydrous ethanol + 1/2 xylene. XI. Dehydration, transparency, sealing After dehydration with the above ethanol series, transparent with xylene, seal the film with Canada resin or neutral resin. Caveat 1. The purpose of fixation is to kill the tissues or cells quickly, so that their morphology and structure can be kept as close as possible to the state of life. The most commonly used fixative is FAA or FPA (formaldehyde: propionic acid: 70% ethanol = 1:1:18, V/V). For delicate samples with high water content, 50% ethanol can be used instead of 70% ethanol. The amount of fixative is usually 50 times that of the sample. 2. The material should not be left in high concentration of ethanol for too long, because ethanol will harden the material, and the material will become brittle and fragile when sliced. 3. Xylene not only removes ethanol, but also has a transparent effect on the material, so it is also known as a transparent agent. 4、When putting the broken wax block, there should be a piece of paper between the material and the wax, so as to avoid direct and immediate contact between the wax and the material, which will cause the material to shrink. 5、During the process of slicing and dividing the wax block, try to make the cut surface of the material and the knife perpendicular. 6、Before sticking the slices, the wax tape must be covered well to prevent the dust from entering into the inside, if the dust is stuck on the slices, it will make the structure on the slices made in the future fuzzy. 7， stained paraffin sections of more staining methods, can be selected according to the purpose of the study. For more product details, please visit Aladdin Scientific website.
Reagents:
① plant material
② fixative (commonly used FAA or FPA)
③ Various grades of ethanol (concentration depends on the sample), xylene solution, anhydrous ethanol-xylene mixture
④ Distilled water
⑤ Paraffin block
⑤ Wax block ⑥ Adhesive
⑦ Fuchsia and solid green dyes
⑧ Canada gum or neutral gum