CN104374601A - Mature grain seed resin section making method - Google Patents
Mature grain seed resin section making method Download PDFInfo
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Abstract
The invention discloses a mature grain seed resin section making method. The method includes the steps of selecting materials, fixing, rinsing, dewatering, performing resin permeation, embedding, polymerizing, blocking, making semi-thin sections, staining and the like. The method is characterized in that firstly, a 4% paraformaldehyde and 2.5% glutaraldehyde stationary liquid secondary fixing technique is adopted; secondly, LR White resin which is low in viscosity and pretty high in infiltration capability is used as an embedding medium; thirdly, a mature seed power cutting technique is set up; fourthly, a 45-60 DEG C secondary section flatting method is set up. The mature grain seed resin section making method has the advantages that the defects of severe curling, failure in flatting, proneness to breakage and failure in iodine staining of mature seed sections made according to conventional methods are overcome, mature grain seed resin sections which are evident in contrast and complete and clear in tissue and cell structure can be obtained, and accordingly study on structures and functions of mature seeds is accurately technically supported.
Description
Technical field
The present invention relates to microscopic tissue sections technical field, particularly relate to a kind of resin slicer method for making of kernel maturity seed.
Background technology
Cereal comprises the grasses such as paddy rice, wheat, corn, and their seed is the main source of grain, feed and the raw material of industry.In cereal, endosperm is the important component part of seed, and its main reserve substance is starch and albumen.Starch accumulates in amyloplaste, and storage protein accumulates in proteosome.In cereal seed, the content of starch, albumen and ratio determine the nutritive value of cereal, have material impact to the processing of cereal and utilization, therefore study starch in seed endosperm and albumen has great importance to the quality and yield improving seed.
Kernel maturity seed is used in a large number at food, feed and industrial circle, and at present the research of cereal seed morphosis is mainly concentrated on to the stage of development of seed, also report is rarely had to the research of mature seed, mainly due to: 1. along with seed growth and grouting enrich, content of starch raises gradually, seed is hardening gradually, and embedding medium is difficult to infiltration, causes cutting into slices easily broken; 2. traditional dicing method needs to make tank and extends piece and drag for sheet, and mature seed due to inside finer and close, section can severe curl, cannot launch in tank; 3. traditional dicing method cannot cut complete mature seed section, can only cut scrappy region, brings difficulty to the areal distribution of each composition in research seed.Therefore need badly and existing microsection manufacture method is improved and optimized, invent a kind of microsection manufacture method of kernel maturity seed.
Summary of the invention
In order to overcome the deficiency of above-mentioned existing microsection manufacture method, the invention provides a kind of resin slicer method for making of kernel maturity seed.
The technical solution adopted in the present invention is as follows:
(1) draw materials: choose the normal kernel maturity seed of particle shape, carefully peel off clever shell, take out complete caryopsis for subsequent use;
(2) fixing:
1. 4% paraformaldehyde-2.5% glutaraldehyde immobile liquid is prepared: 100mL 0.2mol/L phosphate buffer (p H 7.4)+80mL10% paraformaldehyde aqueous solution+20mL 25% glutaraldehyde water solution;
2. be immersed in immobile liquid by got caryopsis, the consumption of immobile liquid is 40 times of sample volume, is put in 4 DEG C of refrigerators and fixes 12h, makes caryopsis softening and preliminary fixing;
3. open cross-section from middle part for softening caryopsis with sharp blade again, cut the tissue block that 1mm in the middle part of caryopsis is thick, be again soaked in immobile liquid, be put in 4 DEG C of refrigerators and fix 48h, make tissue and cell fully fixing.
(3) rinsing: suck immobile liquid, the caryopsis after fixing with the rinsing of 0.1M phosphate buffer, altogether rinsing 5 times, each 30min, fully washes away immobile liquid.
(4) dewater: compound concentration is the ethanol water of 30%, 50%, 70% and 90%, dewaters step by step to caryopsis according to the gradient of 30%-50%-70%-90%-100%-100%, every grade of 15min.
(5) resin infiltration:
1. prepare London white glue (LR W hite) resin: 40mL resin+0.8g catalyzer (Benzoyl Peroxide), magnetic agitation makes abundant dissolving, uses after being put in 4 DEG C of refrigerator 24h;
2. compound concentration is the resin ethanolic solution of 25%, 50% and 75%: resin and absolute ethyl alcohol form according to the ratio mixture of 1:3,1:1 and 3:1;
3. according to the gradient of 25%-50%-75%-100%-100%, caryopsis is permeated step by step, every grade of 12h.
(6) embed: the caryopsis transversal section permeated is put in imbedded mold down, slowly inject pure resin submergence caryopsis, avoid occurring bubble.
(7) be polymerized: be placed on by imbedded mold in baking oven, 60 DEG C of polymerizations 1-2 days (time must control within 2 days, prevents from causing resin shrinkage, causes section difficulty), make hardening of resin.
(8) block is repaiied:
1. first with file, sample surfaces embedding medium is ground off, expose caryopsis.
2. then embedded block is clipped on the specimen holder of microtome, 2mm is exposed on top, to prune embedding medium with the angle at 45 ° with surface level by the surrounding of sharp blade at caryopsis under stereomicroscope, cone-shaped body is accomplished in its side, and upper polygon parallel is below accomplished in the front of sample.
(9) semithin section:
1. mounting glass cutter, is placed in cutter holder and clamps;
2. adjust the position of embedded block and glass cutter, tool setting under stereoscope, make the knife edge and sample in the face of neat:
Whether a) open the backlight of bottom, close other illuminating lamps, feed makes sample face and the knife edge close, can see an appearance bright wisp on sample face, utilize this bright wisp judgement sample to align with cutter;
B) first adjust upper and lower two limits, sample face parallel with the knife edge: whether two limits up and down observing bright wisp are parallel, if not parallel, then adjust specimen holder and rotate knob, make it parallel.
C) left and right adjusting sample face is again parallel with the knife edge: whether the limit, two, left and right observing bright wisp is isometric, if Length discrepancy, then adjusts the knife edge and rotates knob, make it equal.
D) finally adjust the parallel with the knife edge up and down of sample face: move up and down sample face, about observation bright wisp, whether the length on two limits changes, if change, then adjusts sample face and rotates knob, make its length constant;
3. after tool setting completes, feed make the knife edge and sample close, sample face lower edge and the knife edge contour, rotate sample arm lifting button sample arm is moved up and down, till making the knife edge just switch to tissue.
4. slice thickness is set to 2 microns, carries out hand section: the right hand slowly rotates sample arm lifting button and starts section, and in the whole process of section, left hand takes eyelash pencil to be placed on gently directly over section front edge, prevents it curling.This process is the most key, and left hand will remain stable, prevents eyelashes from encountering the knife edge or poking section;
5. get clean microslide, drip distilled water, carefully clamp slicing edge with tweezers, by section to be placed on gently on water droplet with surface level angle at 45 °, dry on 45 DEG C of heating plates, section is flattened.
6. be put in baking oven by section, 60 DEG C of 12h, prevent flake in dyeing course.
(10) dye:
1. iodine liquid preparation: 5g iodine+10g potassium iodide+85mL distilled water, dilutes 80 times as dye liquor with 50% glycerine after dissolving completely.
2. sarranine-methyl violet dye liquor preparation: a) sarranine dye liquor: 1g sarranine+10mL 95% ethanol dissolves, then is settled to 100mL; B) methyl violet dye liquor: 0.5g methyl violet+100mL distilled water.
3. coomassie brilliant blue R250 dye liquor preparation: 1g coomassie brilliant blue R250+7% aqueous acetic acid, 60 DEG C of dissolvings.
4. iodine staining (dye starch, in bluish violet): after dripping iodine liquid, lucifuge leaves standstill 10min, with basis of microscopic observation after cover glass mounting;
5. sarranine-methyl violet staining (dye structure):
A) section is put on heating plate and is heated to 55 DEG C;
B) sarranine dye liquor dyeing 12min is dripped;
C) wash 5min, heating plate is dried;
D) methyl violet dye liquor dyeing 5min is dripped;
E) 5min is washed, basis of microscopic observation after heating plate is dried.
6. coomassie brilliant blue R250 dyeing (dye albumen, in mazarine):
A) section is put on heating plate and is heated to 55 DEG C;
B) 7% acetic acid process 5min is dripped;
C) coomassie brilliant blue R250 dye liquor dyeing 10min is dripped;
D) 7% acetic acid is dripped, color separation 2min;
E) 5min is washed, basis of microscopic observation after heating plate is dried.
Cut into slices after the dyeing of iodine liquid, sarranine-methyl violet dye liquor or coomassie brilliant blue R250 dye liquor, observe under an optical microscope, the image that resolution is high, contrast is moderate can be obtained.
Compared with existing microsection manufacture method, advantage of the present invention is:
(1) the resin slicer method for making being applicable to various types of grain mature seed is established.
(2) have employed 4% paraformaldehyde-2.5% glutaraldehyde immobile liquid secondary technique for fixing, first carry out softening to whole seed and pre-fix, then seed is cut thin after again fix, the eucaryotic cell structure of mature seed can be preserved so in good condition.
(3) employ that viscosity is very low, penetrating power is very strong, and the resin London white glue that water wettability is also very strong (LR W hite) is as embedding medium, it may be used for very large, the inner very fine and close mature seed embedding of volume, and facilitate various water soluble dyestuffs to enter, Color is splendid, very easy to use.
(4) establish the dry incision technology of a kind of mature seed, without the need to making tank in slicing processes, directly can carry out dry cutting to embedded block, simplifying step.
(5) intactly can cut the whole section of caryopsis, overcome conventional method and can make section severe curl, sheet cannot be opened up and hold breakable shortcoming.
(6) establish 45 DEG C of-60 DEG C of secondarys exhibition sheet methods, mature seed section bonds firmly with microslide, can not flake in dyeing course.
(7) section that the method is made can carry out specific stain with iodine liquid to amylum body, and Epon812, Spurr ' section of making of s resin cannot dye with iodine liquid, can only dye by PAS (periodic acid-Schiffreagent) method to amylum body.The amylum body of PAS method dyeing is easily out of shape, and sharpness and contrast are all very low, be unfavorable for the observation of starch granule structure, and the starch granule structure of iodine staining are clear, and contrast is obvious, is more conducive to observe.
Accompanying drawing explanation
Fig. 1 is the shape that rice paddy seed embedded block repaiies block rear surface;
Fig. 2 is the schematic diagram of slicing processes committed step, wherein, and A: eyelashes; B: section front end; C: the glass knife edge; D: embedded block front;
Fig. 3 is the microphoto of the ripe rice paddy seed section of iodine staining, wherein, and 1: amyloplaste; 2: amylum body;
Fig. 4 is the microphoto of the ripe rice paddy seed section of sarranine-methyl violet staining, wherein, and 1: amyloplaste; 2: amylum body; 3: cell membrane;
Fig. 5 is the microphoto that coomassie brilliant blue R250 is coloured to the section of boiled water rice, wherein, and 4: proteosome.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
Embodiment 1:
(1) draw materials: take off Mature seed of rice from spike of rice, carefully peel off clever shell with dissecting needle, take out complete caryopsis, select 10 normal caryopsis of particle shape and be put in penicillin bottle for subsequent use;
(2) fixing:
1. 0.2mol/L phosphate buffer (p H 7.4) is prepared:
A) mother liquor A (0.2mol/L sodium hydrogen phosphate aqueous solution): take 71.64g Na2HPO412H2O, be settled to 1000mL with distilled water;
B) mother liquor B (0.2mol/L biphosphate sodium water solution): take 31.21g Na H2PO42H2O, be settled to 1000mL with distilled water;
C) according to A liquid: the ratio mixing of B liquid=81:19, regulates p H to 7.4.
2. 10% paraformaldehyde aqueous solution is prepared:
A) take 20g paraformaldehyde in 250mL triangular pyramidal bottle, add 200mL distilled water, put into the magnetic stir bar of 25*9mm specification;
C) 0.1mol/L sodium hydrate aqueous solution is prepared: take 40mg NaOH in 10mL centrifuge tube, add 10mL distilled water, mix;
D) in conical flask, dropwise drip 0.1mol/L sodium hydrate aqueous solution just to clarify to paraformaldehyde aqueous solution;
E) be cooled to room temperature, regulate p H to 7.4.
3. prepare 4% paraformaldehyde-2.5% glutaraldehyde immobile liquid: 100mL 0.2mol/L phosphate buffer (p H 7.4)+80mL10% paraformaldehyde aqueous solution+20mL 25% glutaraldehyde water solution fully mixes, regulate p H to 7.4.
4. in penicillin bottle, add 10mL 4% paraformaldehyde-2.5% glutaraldehyde immobile liquid, make caryopsis be immersed in immobile liquid, be put in 4 DEG C of refrigerators and fix 12h, make caryopsis softening and preliminary fixing;
5. take out caryopsis with tweezers, open cross-section from middle part for softening caryopsis with sharp blade, cut the tissue block that 1mm in the middle part of caryopsis is thick, be again soaked in the immobile liquid in penicillin bottle, be put in 4 DEG C of refrigerators and fix 48h, make tissue and cell fully fixing.
(3) rinsing:
1. 0.1mol/L phosphate buffer (p H 7.4) is prepared: 100mL 0.2mol/L phosphate buffer+100mL distilled water, fully mixes.
2. carefully suck immobile liquid with clean suction pipe, do not encounter caryopsis section, then the caryopsis after fixing with the rinsing of 0.1mol/L phosphate buffer, altogether rinsing 5 times, each 30min, fully washes away immobile liquid.
(4) dewater: compound concentration is the ethanol water of 30%, 50%, 70% and 90%, dewaters step by step to caryopsis according to the gradient of 30%-50%-70%-90%-100%-100%: every grade of 10mL, dehydration 15min.
(5) resin infiltration:
1. London white glue (LR W hite) resin is prepared: measure 40mL resin in 50mL centrifuge tube, taking 0.8g catalyzer (Benzoyl Peroxide) pours in centrifuge tube, magnetic agitation 20min makes abundant dissolving, uses after being put in 4 DEG C of refrigerator 24h;
2. compound concentration is the resin ethanolic solution of 25%, 50% and 75%: resin and absolute ethyl alcohol form according to the ratio mixture of 1:3,1:1 and 3:1;
3. according to the gradient of 25%-50%-75%-100%-100%, caryopsis is permeated step by step: every grade of 5mL, be put in 4 DEG C of refrigerators, infiltration 12h.
(6) embed: clamp caryopsis side gently with tweezers, transversal section is put in imbedded mold down, slowly inject pure resin submergence caryopsis with 200mL liquid-transfering gun, avoid occurring bubble.
(7) be polymerized: be placed on by imbedded mold in baking oven, 60 DEG C of polymerizations 1-2 days (time must control within 2 days, prevents from causing resin shrinkage, causes section difficulty), make hardening of resin.
(8) block is repaiied:
1. first with file, sample surfaces embedding medium is ground off, expose caryopsis.
2. then embedded block is clipped on the specimen holder of microtome, 2mm is exposed on top, and to prune embedding medium with the angle at 45 ° with surface level by the surrounding of sharp blade at caryopsis under stereomicroscope, cone-shaped body is accomplished in its side, upper hexagon parallel is below accomplished in the front of sample, as shown in Figure 1.
(9) semithin section:
1. mounting glass cutter, is placed in cutter holder and clamps;
2. adjust the position of embedded block and glass cutter, tool setting under stereoscope, make the knife edge and sample in the face of neat:
Whether a) open the backlight of bottom, close other illuminating lamps, feed makes sample face and the knife edge close, can see an appearance bright wisp on sample face, utilize this bright wisp judgement sample to align with cutter;
B) first adjust upper and lower two limits, sample face parallel with the knife edge: whether two limits up and down observing bright wisp are parallel, if not parallel, then adjust specimen holder and rotate knob, make it parallel;
C) left and right adjusting sample face is again parallel with the knife edge: whether the limit, two, left and right observing bright wisp is isometric, if Length discrepancy, then adjusts the knife edge and rotates knob, make it equal;
D) finally adjust the parallel with the knife edge up and down of sample face: move up and down sample face, about observation bright wisp, whether the length on two limits changes, if change, then adjusts sample face and rotates knob, make its length constant;
3. after tool setting completes, feed make the knife edge and sample close, sample face lower edge and the knife edge contour, rotate sample arm lifting button sample arm is moved up and down, till making the knife edge just switch to tissue.
4. slice thickness is set to 2 microns, carry out hand section: the right hand slowly rotates sample arm lifting button and starts section, in the whole process of section, as shown in Figure 2, the front D of embedded block at the uniform velocity, lentamente moves down, glass knife edge C cuts section gradually, and left hand takes eyelash pencil to be placed on gently directly over section B edge, front end, prevents it curling.This process is the most key, and left hand will remain stable, prevents eyelashes A from encountering the knife edge or poking section;
5. get clean microslide, drip distilled water, carefully clamp slicing edge with tweezers, by section to be placed on gently on water droplet with surface level angle at 45 °, dry on 45 DEG C of heating plates, section is flattened.
6. be put in baking oven by section, 60 DEG C are dried 12h, prevent flake in dyeing course.
(10) dye:
1. iodine liquid preparation: 5g iodine+10g potassium iodide+85mL distilled water, dilutes 80 times as dye liquor with 50% glycerine after dissolving completely.
2. sarranine-methyl violet dye liquor preparation:
A) sarranine dye liquor: 1g sarranine+10mL 95% ethanol dissolves, then is settled to 100mL;
B) methyl violet dye liquor: 0.5g methyl violet+100mL distilled water.
3. coomassie brilliant blue R250 dye liquor preparation: 1g coomassie brilliant blue R250+7% aqueous acetic acid, 60 DEG C of dissolvings.
4. iodine staining (dye starch, in bluish violet): after dripping iodine liquid, lucifuge leaves standstill 10min, with basis of microscopic observation after cover glass mounting;
5. sarranine-methyl violet staining (dye structure):
A) section is put on heating plate and is heated to 55 DEG C;
B) sarranine dye liquor dyeing 12min is dripped;
C) wash 5min, heating plate is dried;
D) methyl violet dye liquor dyeing 5min is dripped;
E) 5min is washed, basis of microscopic observation after heating plate is dried.
6. coomassie brilliant blue R250 dyeing (dye albumen, in mazarine):
A) section is put on heating plate and is heated to 55 DEG C;
B) 7% acetic acid process 5min is dripped;
C) coomassie brilliant blue R250 dye liquor dyeing 10min is dripped;
D) 7% acetic acid is dripped, color separation 2min;
E) 5min is washed, basis of microscopic observation after heating plate is dried.
Cut into slices after the dyeing of iodine liquid, sarranine-methyl violet dye liquor or coomassie brilliant blue R250 dye liquor, observe under an optical microscope, the image that resolution is high, contrast is moderate can be obtained, as Fig. 3, Fig. 4 and Fig. 5.Fig. 3 is for section is through iodine staining, and demonstrate complete in rice-embryo milk cell and amylum body 2 clearly, multiple polygon amylum body 2 rearranges amyloplaste 1.Fig. 4 for section is through the dyeing of sarranine-methyl violet dye liquor, show clearly cell membrane 3, amyloplaste 1 and amylum body 2, Fig. 5 for section is through the dyeing of coomassie brilliant blue R250 dye liquor, complete and proteosome 4 clearly in display albuminous cell.
Claims (10)
1. a resin slicer method for making for kernel maturity seed, is characterized in that, comprise the following steps:
(1) draw materials: kernel maturity seed, peel off clever shell, take whole caryopsis;
(2) fixing: got caryopsis is immersed in 4% paraformaldehyde--24 h in 2.5% glutaraldehyde immobile liquid, make caryopsis soften; Opening cross-section for softening caryopsis, cut the tissue block that in the middle part of caryopsis, 1 mm is thick, be again soaked in 4% paraformaldehyde--48 h in 2.5% glutaraldehyde immobile liquid, make tissue and cell fully fixing;
(3) rinsing: the caryopsis after fixing with 0.1 M phosphate buffer rinsing, fully washes away immobile liquid;
(4) dewater: with 30%-50%-70%-90%-100%-100% ethanol, caryopsis is dewatered step by step, every grade of 15 min;
(5) resin infiltration: with 25%-50%-75%-100%-100% London white glue resin, caryopsis is permeated step by step, every grade of 12 h;
(6) embed: the caryopsis permeated is put in imbedded mold, injects pure resin;
(7) be polymerized: be placed on by imbedded mold in baking oven, 60 DEG C of polymerization 1-2 days, make hardening of resin;
(8) block is repaiied: craft is carried out to embedded block and repaiies block, pruned by Excess resin around caryopsis;
(9) semithin section: cutting thickness with ultramicrotome is 2 micron sections, transfers to section on the water droplet on microslide, is placed on by microslide on 45 DEG C of heating plates dry, section is flattened;
(10) dye: the section of flattening, through the dyeing of iodine liquid, sarranine-methyl violet dye liquor or coomassie brilliant blue R250 dye liquor, is observed under an optical microscope, can be obtained the image that resolution is high, contrast is moderate.
2. microsection manufacture method according to claim 1, it is characterized in that, in step (2), the formula of described immobile liquid is: 100 mL 0.2mol/L pH 7.4 phosphate buffers, 80 mL 10% paraformaldehyde aqueous solution and 20 mL 25% glutaraldehyde water solutions.
3. microsection manufacture method according to claim 1, is characterized in that, in step (2), the consumption of described immobile liquid is 40 times of sample volume, and fixing temperature is 4 DEG C.
4. microsection manufacture method according to claim 1, is characterized in that, in step (5), described 25%-50%-75% resin is that 100% London white glue resin and 100% ethanol form by the volume ratio mixture of 1:3,1:1 and 3:1 respectively.
5. microsection manufacture method according to claim 1, is characterized in that, in step (6), must keep section down, slowly must add, prevent bubble when adding pure resin during described caryopsis embedding.
6. microsection manufacture method according to claim 1, is characterized in that, in step (8), described in repair block detailed process be:
1. first with file, sample surfaces embedding medium is ground off, expose caryopsis;
2. then embedded block is clipped on the specimen holder of microtome, 2mm is exposed on top, to prune embedding medium to become the angle of 45 with surface level by the surrounding of sharp blade at caryopsis under stereomicroscope, cone-shaped body is accomplished in its side, and upper polygon parallel is below accomplished in the front of sample.
7. microsection manufacture method according to claim 1, is characterized in that, in step (9), in the dicing method of described semithin section, directly carries out dry cutting to embedded block, intactly cuts the whole section of caryopsis.
8. microsection manufacture method according to claim 1, is characterized in that, in step (9), the dicing method of described semithin section is:
1. mounting glass cutter, is placed in cutter holder and clamps;
2. adjust the position of embedded block and glass cutter, tool setting under stereoscope, make the knife edge and sample in the face of neat:
Whether a) open the backlight of bottom, close other illuminating lamps, feed makes sample face and the knife edge close, can see an appearance bright wisp on sample face, utilize this bright wisp judgement sample to align with cutter;
B) first adjust upper and lower two limits, sample face parallel with the knife edge: whether two limits up and down observing bright wisp are parallel, if not parallel, then adjust specimen holder and rotate knob, make it parallel;
C) left and right adjusting sample face is again parallel with the knife edge: whether the limit, two, left and right observing bright wisp is isometric, if Length discrepancy, then adjusts the knife edge and rotates knob, make it equal;
D) finally adjust the parallel with the knife edge up and down of sample face: move up and down sample face, about observation bright wisp, whether the length on two limits changes, if change, then adjusts sample face and rotates knob, make its length constant;
3. after tool setting completes, feed make the knife edge and sample close, sample face lower edge and the knife edge contour, rotate sample arm lifting button sample arm is moved up and down, till making the knife edge just switch to tissue;
4. slice thickness is set to 2 microns, carries out hand section: the right hand slowly rotates sample arm lifting button and starts section, and in the whole process of section, left hand takes eyelash pencil to be placed on gently directly over section front edge, prevents it curling;
This process is the most key, and left hand will remain stable, prevents eyelashes from encountering the knife edge or poking section;
5. get clean microslide, drip distilled water, carefully clamp slicing edge with tweezers, section is placed on water droplet gently to become 45 jiaos with surface level, dry on 45 DEG C of heating plates, section is flattened;
6. be put in baking oven by section, 60 DEG C of 12h, prevent flake in dyeing course.
9. microsection manufacture method according to claim 1, is characterized in that, in step (10), the formula of described dye liquor is:
1. iodine liquid: 5g iodine+10g potassium iodide+85mL distilled water, dilutes 80 times as dye liquor with 50% glycerine after dissolving completely;
2. sarranine-methyl violet dye liquor:
A) sarranine dye liquor: 1g sarranine+10 mL 95% ethanol dissolves, then is settled to 100 mL;
B) methyl violet dye liquor: 0.5g methyl violet+100 mL distilled water;
3. coomassie brilliant blue R250 dye liquor: 1g coomassie brilliant blue R250+7% aqueous acetic acid, 60 DEG C of dissolvings.
10. microsection manufacture method according to claim 1, is characterized in that, in step (10), described colouring method is:
1. iodine staining: after dripping iodine liquid, lucifuge leaves standstill 10 min, with basis of microscopic observation after cover glass mounting; Starch is bluish violet;
2. sarranine-methyl violet staining:
A) section is put on heating plate and is heated to 55 DEG C;
B) drip sarranine dye liquor to dye 12 min;
C) wash 5 min, heating plate is dried;
D) drip methyl violet dye liquor to dye 5 min;
E) 5 min are washed, basis of microscopic observation after heating plate is dried;
3. coomassie brilliant blue R250 dyeing:
A) section is put on heating plate and is heated to 55 DEG C;
B) 7% acetic acid process 5 min is dripped;
C) drip coomassie brilliant blue R250 dye liquor to dye 10 min; D) 7% acetic acid is dripped, color separation 2 min;
E) wash 5 min, basis of microscopic observation after heating plate is dried, albumen is mazarine.
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| CN108332989B (en) * | 2018-02-27 | 2020-12-29 | 江南大学 | Method for analyzing high-starch-content cereal grains and observing internal structure |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630421A (en) * | 2013-12-03 | 2014-03-12 | 沈阳农业大学 | Production method for paraffin section of paeonia lactiflora mature embryo |
| CN103954484A (en) * | 2014-04-02 | 2014-07-30 | 浙江万里学院 | Method for producing early gonad paraffin section of Chinese soft shell turtle |
-
2014
- 2014-12-08 CN CN201410743579.0A patent/CN104374601B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630421A (en) * | 2013-12-03 | 2014-03-12 | 沈阳农业大学 | Production method for paraffin section of paeonia lactiflora mature embryo |
| CN103954484A (en) * | 2014-04-02 | 2014-07-30 | 浙江万里学院 | Method for producing early gonad paraffin section of Chinese soft shell turtle |
Non-Patent Citations (1)
| Title |
|---|
| 魏玉利 等: ""光合细菌的透射电镜观察"", 《生命科学仪器》 * |
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| CN105067413B (en) * | 2015-09-21 | 2017-07-28 | 河南中医学院 | It is a kind of to be used for the colouring method of the ultra-thin section that tannin is distributed in observation of plant cell |
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| CN110208056A (en) * | 2019-06-06 | 2019-09-06 | 厦门大学附属第一医院 | The tissue chip production method of gastric cancer HER-2 FISH |
| CN117517029A (en) * | 2024-01-05 | 2024-02-06 | 南京农业大学三亚研究院 | Method for making resin slice of wheat mature period grain |
| CN117517029B (en) * | 2024-01-05 | 2024-04-16 | 南京农业大学三亚研究院 | Method for making resin slice of wheat mature period grain |
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