Background and overview[1]
Aniline blue (alcohol-soluble) is a green or reddish-brown crystalline powder, slightly soluble in alcohol, insoluble in water, and turns brown when soluble in concentrated sulfuric acid. After dilution, a blue precipitate is obtained. Preparation method: 4,4′-diaminodiphenylmethane, aniline, aniline hydrochloride, nitrobenzene and ferrous chloride are heated together (170℃) to obtain para-fuchsin, which is then mixed with excess aniline Obtained by heating to 180°C in the presence of benzoic acid. Its use: can be used as dye.
Apply[2]
CN201610422991.1 provides a method for observing callose deposition in creeping bentgrass leaf tissue based on paraffin sectioning and aniline blue fluorescent staining. It is the first time that paraffin sectioning and aniline blue fluorescent staining are used to observe creeping bentgrass leaf tissue. The deposition of callose and the paraffin preparation process took 2 days, which greatly shortened the experimental time for paraffin preparation of creeping bentgrass. It broke through the limitations caused by the material itself and environmental factors, and could complete the paraffin preparation quickly and efficiently. film process.
To achieve the above objectives, the technical solutions adopted by the present invention are:
The method of observing callose deposition in creeping bentgrass leaf tissue based on paraffin sections and aniline blue fluorescence staining method includes the following steps:
S1. Preparation of medicines;
S11. Mix 10ml of formalin, 3ml of acetic acid, 87ml of 50% ethanol, and 5ml of glycerol to obtain FAA fixative;
S12. Mix absolute ethanol and xylene in a ratio of 1:1 to prepare 1/2 xylene;
S13. Weigh 0.1g of water-soluble aniline blue and dissolve it in 100ml of phosphate buffer with a concentration of 0.07mol/L and pH=7 to obtain 0.1% aniline blue fluorescent stain.
S2. Make paraffin sections;
S21. Use a sharp blade to quickly remove the creeping bentgrass leaves with a width of about 0.8mm, cut the leaves into small segments of about 5mm in length, put them into a penicillin vial, add the FAA fixative obtained in step S11, and fix for 12 hours. ;
S22. After being fixed with FAA, perform gradient dehydration in 15%, 30%, 50%, 70%, and 85% ethanol. Dehydrate for 20 min each time, dehydrate in 95% ethanol for 30 min, and dehydrate in absolute ethanol for two seconds. times, 20 minutes each time; then replace 1/2 xylene and xylene in turn to make it transparent, make it transparent in 1/2 xylene for 1 hour, and make it transparent in xylene twice, 30 minutes each time, until the material is completely transparent;
S23. Add crushed wax twice to the penicillin vial containing the obtained transparent material. For the first time, add the same amount of crushed wax as the xylene in the bottle, and place it in an electric blast drying oven at a temperature of 36°C for 1 hour. After the added broken wax is completely melted, add half of the liquid in the bottle, put it in an electric blast drying oven and adjust the temperature to 36°C overnight, then adjust the temperature to 42°C, replace 50% of the paraffin, and keep it for 1 hour, then adjust the temperature to 50°C, replace 75% of the paraffin wax and keep it for 30 minutes, adjust the temperature to 60°C, replace pure paraffin A, pure paraffin B, and pure paraffin C for 30 minutes each, and then use the TKY-BMB paraffin embedding machine for embedding, one wax One material in the block, the size of the wax block is: 0.5cm×0.5cm×0.5cm. Cool the embedded wax block and trim it directly, then slice it or store it in a 4℃ refrigerator for later use;
S24. Use a wheel-type manual microtome to perform continuous slicing with a thickness of 10 μm; adjust the temperature of the HH-6 digital constant temperature water bath to 45°C, add distilled water to a 500ml large beaker and put it into the water bath, and put the cut wax into the water bath. Put the tape into the beaker. After the wax tape is completely unfolded, put the slide coated with egg white and glycerin into the beaker so that the wax tape sticks to the slide. After the slide with the wax tape is dried naturally, Observe the film expansion effect under an optical microscope, and place the film with the best film expansion effect in an electric blast drying oven and bake it at 38°C until the film is completely dry;
S25. Take the dry slices and place them in order with xylene, 1/2 xylene, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, In a vertical dye vat with phosphate buffer and 0.1% aniline blue dye; in xylene and 1/2 xylene for 5 minutes each, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 5S each in 30% ethanol, 10min in phosphate buffer, and 30min in 0.1% aniline blue stain; then take out the stained piece and place it in distilled water for 30S. After washing the impurities on the piece, wait 1/2 Xylene, dimethylbenzene 10S each, after the film is dry, use neutral gum to seal it;
S26. After the obtained film is naturally air-dried, it is observed and photographed under a LeiCaDM6000B fluorescence microscope.
Main reference materials
[1] Compound Dictionary
[2] CN201610422991.1 Method for observing callose deposition in creeping bentgrass leaf tissue based on paraffin sections and aniline blue fluorescence staining
