The method observing the deposition of creeping bentgrass leaf tissue callose based on paraffin section and aniline blue fluorescent staining method

The method observing the deposition of creeping bentgrass leaf tissue callose based on paraffin section and aniline blue fluorescent staining method

  • CN 106,198,465 A
  • Filed: 06/10/2016
  • Published: 12/07/2016
  • Est. Priority Date: 06/10/2016
  • Status: Active Application
First Claim
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1. the deposition process of creeping bentgrass leaf tissue callose is observed based on paraffin section and aniline blue fluorescent staining method, itsIt is characterised by, comprises the steps:

  • S1, the preparation of medicine;

    S11, by formalin 10ml, acetic acid 3ml, ethanol 87ml, 5ml glycerol mix homogeneously of 50%, obtain FAA fixative;

    S12, dehydrated alcohol and dimethylbenzene are hybridly prepared into 1/2 dimethylbenzene with the ratio of 1;


    S13, weigh 0.1g water-soluble aniline blue and be dissolved in the phosphate buffer that 100ml concentration is 0.07mol/L, pH=7,The aniline blue fluorescent dye of 0.1%.S2, making paraffin section;

    S21, quickly take off the creeping bentgrass blade of width about 0.8mm with sharp blade, blade cuts is become length about 5mmThe segment of left and right, puts in penicillin bottle, adds the FAA fixative of step S11 gained, fixing 12h;

    S22, after FAA is fixing, replace successively and the ethanol of 15%, 30%, 50%, 70%, 85% carry out serial dehydration, each de-Water 20min, is dehydrated 30mim in 95% ethanol, is dehydrated twice, each 20min in dehydrated alcohol;

    Replace 1/2 diformazan the most successivelyBenzene, dimethylbenzene carry out transparent, transparent 1h in 1/2 dimethylbenzene, in dimethylbenzene transparent twice, and each 30min, until material is completeTill transparent;

    S23, in equipped with the penicillin bottle of gained transparent material, add broken wax twice, add for the first time and dimethylbenzene equivalent in bottleBroken wax, put in electric drying oven with forced convection after temperature 36 DEG C keeps 1h to melt completely to added broken wax, add liquid one in bottleThe broken wax of half, put into electric drying oven with forced convection adjusts the temperature to 36 DEG C overnight after, adjust the temperature to 42 DEG C, replace 50% paraffinAfter keeping 1h, temperature is adjusted to 50 DEG C, after the paraffin of displacement 75% keeps 30min, adjusts the temperature to 60 DEG C, replaces paraffin refined wax successivelyA, paraffin refined wax B, each 30min of paraffin refined wax C, TKY-BMB type paraffin wax embedding embeds, a material, wax stone in a wax stoneSize be;

    0.5cm ×

    0.5cm ×

    0.5cm, after embedded wax stone cooling, directly repair block, carry out cutting into slices or be saved in 4 DEG CRefrigerator is standby;

    S24, employing wheel type manual microtome carry out serial section, thickness 10 μ


    HH-6 digital display water bath with thermostatic control pot temperature is regulatedTo 45 DEG C, add distilled water at 500ml large beaker and put in water-bath, the wax band light cut is faced down and puts in beaker, treatAfter wax band is fully deployed, the microscope slide that painting wipes Ovum Gallus domesticus album glycerol stretches in beaker, makes wax band adhere on microscope slide, will be stained with waxAfter the microscope slide of band dries naturally, observe exhibition sheet effect under an optical microscope, take the effective slice, thin piece of exhibition sheet and be placed in electric heating drumWind drying baker toasts at 38 DEG C, until slice, thin piece is completely dried;

    S25, take dry slice, thin piece, be put into successively respectively added with dimethylbenzene, 1/2 dimethylbenzene, the ethanol of 95%, the ethanol of 85%,The ethanol of 70%, the ethanol of 50%, the ethanol of 30%, phosphate buffer, 0.1% aniline blue stain vertical dye vat in;

    In dimethylbenzene, 1/2 dimethylbenzene each 5min, the ethanol of 95%, the ethanol of 85%, the ethanol of 70%, the ethanol of 50%, 30%30min in the aniline blue stain of each 5S in ethanol, 10min in phosphate buffer, 0.1%;

    Then the slice, thin piece after dyeing is takenGo out, be placed in 30S in distilled water, after cleaning the impurity on slice, thin piece, cross 1/2 dimethylbenzene, each 10S of dimethylbenzene, treat that slice, thin piece is adopted after dryingUse neutral gum mounting;

    S26, by after the slice, thin piece natural air drying of gained, be placed in LeiCaDM6000B fluorescence microscopy Microscopic observation and take pictures.

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