CN105547792A - Method for simultaneously detecting spermatoblast apoptosis and membrane integrity - Google Patents
Method for simultaneously detecting spermatoblast apoptosis and membrane integrity Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
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Abstract
The invention discloses a method for simultaneously detecting spermatoblast apoptosis and membrane integrity. The method comprises the steps that a lower osmatic solution is prepared, 735 mg of sodium citrate and 1350 mg of fructose are added into every 100 ml of distilled water for forming the lower osmatic solution, the ion strength of the lower osmatic solution is made to be 0.15, and the osmotic pressure of the solution is 150 morse moores. The method can solve problems existing in the prior art, the method has the advantages that whether the membrane of a sperm is complete or not and whether chromatins of the sperm are complete or not can be displayed at the same time, nontoxicity and non-carcinogenicity pachymaran is used for replacing an original 4% paraformaldehyde stationary solution, the sperm is kept in the swelling or non-swelling form after being processed in the previous step, and harmful chemicals can be prevented from polluting environment and injuring experimenters; agar gel is used for enabling the sperm to be well spread on a glass slide, the sperm can be kept in the swelling or non-swelling form after being processed in the previous steps, and the quality of the sperm can be accurately judged in an assisting mode.
Description
Technical field
The present invention relates to the detection method that clinical medicine uses, is exactly a kind of method simultaneously detecting spermatoblast apoptosis and film integrality.
Background technology
Sperm membrane integrity and sperm metabolism, acrosome reaction, capacitation and sperm-egg fusion closely related, measure whether sperm membrane complete can reflect sperm function exactly and predict the potential fertility of sperm.Since setting up sperm tail hypotonic swelling measuring Human sperm membrane function from 1984, the experiment of sperm tail hypotonic swelling has become one of detection method of few at present several evaluation sperm membrane functions, in the fundamental research applying to male genetic contraception more and more widely and sterile clinical diagnosis and treatment.The mass exchange of cell and metabolic activity rely on cell membrane to carry out, sperm is very easily subject to various chemical substance in man, female genital tract, sperm membrane directly affects vigor and the metabolism of sperm to the transmission capacity of these materials, thus can be related to capacitation, complete the overall process of fertilization.Therefore, sperm membrane function is evaluated significant in sperm function detects.But, simple sperm hypo-osmotic swelling test experiment can only determine that whether a sperm membrane is complete, and its DNA and chromatin whether complete (apoptosis) can not be differentiated, if only detect merely the integrality of sperm membrane, and do not detect the activity that Sperm Apoptosis can't judge sperm exactly, therefore, existing experimental technique is difficult to accurately to judge whether the film of same sperm and DNA and chromatin damage and injures apoptosis, is namely difficult to make accurate evaluation to the quality of sperm.
Summary of the invention
Object of the present invention, there is provided a kind of method simultaneously detecting spermatoblast apoptosis and film integrality, it can solve now above-mentioned technology Problems existing, simultaneously its advantage to show whether its film of same sperm is complete and whether chromatin is complete, and replace original 4% paraformaldehyde immobile liquid with pachymaran liquid that is non-toxic and carcinogenicity, the form of the swelling after the process of sperm maintenance previous step or not swelling can be made, and harmful chemicals thing environmental pollution can be prevented and experimenter is caused damage.Use agar gel that sperm can be enable well to spread out on microslide, enable sperm keep the swelling after step process or pneumonedema bulging state constant, people can be assisted accurately to judge the quality of sperm.
The object of the invention is to be achieved through the following technical solutions: a kind of method simultaneously detecting spermatoblast apoptosis and film integrality, it is characterized in that: operation step is poly-as follows: 1. prepare hypotonic medium: in every 100 ml distilled waters, put into 735 milligrams of sodium citrates and 1350 milligrams of fructose make hypotonic medium, and make its ionic strength be 0.15(coefficient), osmotic pressure 150 rubs the solution of this mole; 2. to every 1 milliliter of step 1. described in hypotonic medium in add 0.1 milliliter of seminal fluid, mixing, in water temperature be in the water of 37 DEG C water-bath after 30 minutes seminal fluid after Hypotonic treatment, observe 200 sperms, the sperm ratio of calculating afterbody swelling; 3. get 1 milliliter of step 2. described in Hypotonic treatment after seminal fluid, with centrifugal treating under the centrifuge speed of 1000 revs/min 5 minutes, after stratification, abandon supernatant, gained seminal fluid sperm sediment phosphate buffer (PBS) is diluted to sperm concentration 1,000 ten thousand/milliliter, obtains dilution seminal fluid; 4. get step 3. described in dilution seminal fluid 0.2 milliliter and agar gel by volume 1:1 ratio mix, be placed in the centrifuge tube of 1.5 milliliters, hatch under room temperature 37 DEG C of conditions and must configure seminal fluid in 5 minutes; 5. aspiration step 4. described in configuration seminal fluid 50 microlitre insert on microslide, add a cover cover glass, under the condition of environment temperature 4 DEG C place 4 minutes; Described microslide is the microslide that surface is covered with the poly-D-lysine of thin layer; 6. the cover glass on microslide 5. described for step is removed, microslide to be immersed in pachymaran liquid 25 minutes, with the process that fixes to material on microslide; Pachymaran liquid forms by adding 1 gram of pachymaran configuration in every 10 ml physiological salines; 7. use phosphate buffer (PBS) embathe step 6. described fixing after microslide secondary, each 5 minutes; Then in the TritonX-100 of 10 milliliter 0.2%, embathing process 5 minutes by embathing rear microslide, embathing secondary with phosphate buffer more afterwards, each 5 minutes; 8. mixed liquor is prepared with 50 microlitre TdT and the fluorescein-labeled dUTP liquid mixing of 450 microlitres; 7. the several piece microslide after processing through step is divided into negative control group and positive controls, and the every block microslide wherein to negative control group adds the fluorescein-labeled dUTP liquid of 50 microlitre; Every block microslide of positive controls first adds 100 microlitre DNase1, reacts at 15 ~ 25 DEG C × 10 minutes, is processed by every block microslide afterwards, embathe 5 minutes, then embathe secondary with PBS in 10 milliliters in the TritonX-100 of 0.2%, each 5 minutes; Reaction 37 DEG C × 30 minutes in dark wet box after all microslides of negative control group and positive controls are dry, PBS rinsing 3 times, each 5 minutes; Dark wet box is airtight lucifuge, the humidity box more than 80%; 9. all microslides after step 8. finally being processed add 50 ~ 100 microlitre diaminobenzidine (DAB) substrates, react 15 ~ 25 DEG C × 10 minutes; 10. the microslide haematoxylin after step 9. being processed or methyl green counterstain, use tap water after 5 seconds immediately; Gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting; Examine under a microscope result.
For realizing object of the present invention further, can also realize by the following technical solutions: step 1. described in distilled water be distilled water.Step
described in microscope be that Germany produces Leica board DM4000B type microscope.Step 9. described in diaminobenzidine (DAB) substrate be diaminobenzidine developer, add rear natural light basis of microscopic observation sperm.The extracting method of described pachymaran is: the grinding of 1000 grams, herbal raw material Poria cocos, cross 60 mesh sieves, be placed in 1000 milliliters, the physiological saline of 0.9% and soak 1 hour, the ratio of Poria cocos raw material and physiological saline is 1:1(weight ratio), be placed in the Chinese medicine extraction machine of 100 ~ 120 DEG C and add extraction heat 30 minutes, after taking out, filter and remove residue obtains Poria cocos extract, and the 1000 milliliters of 60 DEG C of dryings of Poria cocos extract are obtained rough pachymaran 320 grams in 2 hours; Take 100 grams of rough pachymarans to put in 500 milliliters of round-bottomed flasks, add sherwood oil 250 milliliters in 60 ~ 90 DEG C of backflows degreasing in 1 hour, filter, sherwood oil reclaims to obtain filter residue; Filter residue volatilizes after solvent with 80% ethanol, 250 milliliters of steeped overnight, 80 ~ 90 DEG C of refluxing extraction 2 times, each 1.5 hours, filter to obtain the dregs of a decoction, dregs of a decoction adding distil water 500 milliliters leaching got 40 minutes in 90 DEG C of warm lixiviates after 1 hour, filtered to obtain extract, again by extract 250 ml distilled water 90 DEG C temperature leaching 30 minutes, filter, merge 40 minutes gained are got in above-mentioned dregs of a decoction adding distil water 500 milliliters leaching in 90 DEG C of warm lixiviates filtrate after 1 hour, be evaporated to 75 milliliters and obtain concentrated filtrate; By concentrated filtrate deproteinization, add activated carbon decolorizing 2 times, suction filtration, filtrate adds 95% ethanol to be made to reach 80%, refrigerator hold over night containing amount of alcohol; Filter to obtain residue, in 60 DEG C of oven dry after residue washs with 95% ethanol, absolute ethyl alcohol, ether, acetone successively, obtain the pachymaran 210 grams of brown, the consumption of activated charcoal is that every 100 milliliters of concentrated filtrates add 1 gram of activated charcoal.With DEAE cellulose column to described pachymaran separation and purification: above DEAE cellulose column once: be packed into by pachymaran in DEAE cellulose column, afterwards, first take off liquid by the polysaccharide liquid that distilled water wash-out obtains a pachymaran component, then carry out gradient elution with 0.2 mol/L sodium hydroxid; Amount to upper DEAE cellulose column six times, each filling pachymaran 2.5 grams, collects the pachymaran eluent of whole six times, dialyses 3 days, must refine pachymaran 10.9 grams of white solids after vacuum freeze drying in flowing water.
Beneficial effect of the present invention is: it can detect spermatoblast apoptosis and film integrality simultaneously, and conclusion has more standby clinical reference value.Measure 32 routine normal fertile men altogether draw and apply sperm hypo-osmotic swelling test and test different conclusions, namely the sperm morphology change utilizing the method for the invention to obtain and apoptosis have 4 types (A-D), wherein A type sperm is afterbody swelling, sperm head dye blueness, namely sperm membrane and chromatin are all complete and apoptosis does not occur, and sperm has mobility or lives; Type B sperm is the sperm of head dye blue (namely TUNEL is normal) and the non-swelling of afterbody (i.e. HOS exception), i.e. complete, the non-apoptosis of Sperm Chromatin and afterbody membrane damage, and we observe this sperm is the sperm having mobility; If use sperm hypo-osmotic swelling test experimental judgment merely, this Type B of result sperm is exactly sperm that is dead or that do not have mobility; And C type sperm is head red colouration or purple (i.e. TUNEL exception, apoptosis) and the sperm of afterbody swelling (namely HOS is normal), this sperm is just mistaken for movable eupyrene sperm in simple sperm hypo-osmotic swelling test experiment, because sperm uropatagium is swelling, namely complete; D type sperm is head red colouration or purple (namely TUNEL is abnormal, apoptosis) and the sperm of afterbody also not swelling (namely HOS is abnormal), is the sperm of apoptosis and the damage of afterbody film.Our experimental result confirms that the experiment of simple sperm hypo-osmotic swelling test is for judging that the integrality of motility of sperm and film is incomplete, and the Nick End labelling experiment of simple sperm deoxynucleotide terminal enzyme (DNA) mediation judges whether sperm is also normally incomplete.For A type sperm and D type sperm, simple sperm hypo-osmotic swelling test experiment is consistent with the experimental judgment result of the method for the invention, namely A type sperm is the movable sperm complete with head film-uropatagium, D type sperm is the sperm (apoptosis) that DNA and film all damage, it is dead sperm, but to Type B sperm, simple sperm hypo-osmotic swelling test experiment can only judge that it is Necrospermia, in fact this sperm is sperm of living, and the judgement to C type sperm, simple sperm hypo-osmotic swelling test experiment is then judged as sperm of living, in fact this sperm is Necrospermia, this bright described methods experiment of application just can make accurate judgment to these sperms.The experiment of the method for the invention and HOS/EUNEL Binding experiment, sperm hypo-osmotic swelling test experiment and HOS, the Nick End labelling method that sperm DNA end is transferase mediated and TUNEL.Sperm hypo-osmotic swelling test test (HOS) and the transferase mediated Nick End labelling method (TUNEL) of DNA end are combined on same sperm and carry out, independent HOS and independent TUNEL testing inspection part is made to be that normal sperm is defined as abnormal spermium, because all there is certain defect in two kinds of methods, as HOS test can only detect the integrality of sperm membrane, and DNA integrality and the apoptosis situation of sperm can not be measured, TUNEL test can only measure DNA integrality and the apoptosis situation of sperm, and can not detect the integrality of sperm membrane.
simple HOS applied by table 1, the simple routine normal fertile men monitoring result of TUNEL and HOS/TUNEL Binding experiment 32 compares
hOS/TUNEL combines the simple HOS (%) of (%) simple TUNEL (%)
Pattern1A type 73.1 ± 3.387.2 ± 5.3
*(normal TUNEL) 79.6 ± 4.8 (normal HOS)
Pattern2B type 10.5 ± 1.8
Pattern3C type 4.7 ± 1.2
pattern4D type 11.7 ± 1.7
12.8 ± 2.5
*
(abnormal T UNEL) 20.4 ± 2.3 (abnormal HOS)
* P<0.01 and * * P<0.001, tests with simple HOS and compares.
Accompanying drawing explanation
Fig. 1 is the sperm image that simple TUNEL shows that head dyeing is green (normally) and purple (apoptosis), be labeled as (SCD+), mark 1 in figure and represent the blue rear shown in green eupyrene sperm of head dye, mark 2 is shown as the apoptosis sperm image of purple after representing head dye blueness, not through HOS experiment, afterbody does not have swelling.Microscope magnification: 10 × 100=1000 doubly; Fig. 2 is the image after carrying out HOS/TUNEL Binding experiment, and witness marking 1 dyes the sperm of green and afterbody swelling for A type sperm and head, and mark 2 dyes the sperm of green and non-afterbody swelling for Type B sperm and head.Microscope magnification: 10 × 100=1000 doubly; Fig. 3 is 4 types (A type, Type B, C type and the D type) sperm that the transferase mediated Nick End labelling method Binding experiment of sperm hypo-osmotic swelling test experiment/sperm DNA end and HOS/EUNEL Binding experiment obtain, wherein a figure is display is the A type sperm sperm of afterbody swelling (the head green colouring and), eupyrene sperm; It is Type B sperm (head green colouring and the sperm of non-afterbody swelling) that b figure shows; It is C type sperm that c figure shows, sperm head dye purple (chromatin is imperfect, apoptosis, afterbody swelling), and it is D type sperm that d figure shows, the sperm head dye non-swelling of purple afterbody (apoptosis).
Embodiment
Detect a method for spermatoblast apoptosis and film integrality simultaneously, it is characterized in that: operation step is poly-as follows:
1. prepare hypotonic medium: prepare hypotonic medium: in every 100 ml distilled waters, put into 735 milligrams of sodium citrates and 1350 milligrams of fructose make hypotonic medium, and make its ionic strength be 0.15(coefficient), osmotic pressure 150 rubs the solution of this mole;
2. to every 1 milliliter of step
described in hypotonic medium in add 0.1 milliliter of seminal fluid, mixing, be water-bath after 30 minutes in the water of 37 DEG C in water temperature, obtain seminal fluid after Hypotonic treatment, observe 200 sperms, the sperm ratio of calculating afterbody swelling;
3. get 1 milliliter of step 2. described in Hypotonic treatment after seminal fluid, with centrifugal treating under the centrifuge speed of 1000 revs/min 5 minutes, after stratification, abandon supernatant, gained seminal fluid sperm sediment phosphate buffer (PBS) is diluted to sperm concentration 1,000 ten thousand/milliliter, obtains dilution seminal fluid;
4. get step 3. described in dilution seminal fluid 0.2 milliliter and agar gel by volume 1:1 ratio mix, be placed in the centrifuge tube of 1.5 milliliters, hatch under room temperature 37 DEG C of conditions and must configure seminal fluid in 5 minutes.4. step is diluted seminal fluid 0.2 milliliter and is mixed in 1:1 ratio (volume ratio) with agar gel, be placed in the centrifuge tube of 1.5 milliliters, be in order to sperm well can spread out on microslide, enable the sperm through hypotonic swelling experiment keep its form (swelling or not swelling) constant.Agar gel can be heated to 80 ~ 100 degree of thawings by agar and obtain.
5. aspiration step 4. described in configuration seminal fluid 50 microlitre insert on microslide, add a cover cover glass, under the condition of environment temperature 4 DEG C place 4 minutes; Described microslide is the microslide that surface is covered with the poly-D-lysine of thin layer;
6. the cover glass on microslide 5. described for step is removed, microslide to be immersed in pachymaran liquid 25 minutes, with the process that fixes to material on microslide;
7. use phosphate buffer (PBS) embathe step 6. described fixing after microslide secondary, each 5 minutes; Then in the TritonX-100 of 10 milliliter 0.2%, embathing process 5 minutes by embathing rear microslide, embathing secondary with phosphate buffer more afterwards, each 5 minutes;
8. mixed liquor is prepared with 50 microlitre TdT and the fluorescein-labeled dUTP liquid mixing of 450 microlitres; 7. the several piece microslide after processing through step is divided into negative control group and positive controls, and the every block microslide wherein to negative control group adds the fluorescein-labeled dUTP liquid of 50 microlitre; Every block microslide of positive controls first adds 100 microlitre DNase1, reacts at 15 ~ 25 DEG C × 10 minutes, is processed by every block microslide afterwards, embathe 5 minutes, then embathe secondary with PBS in 10 milliliters in the TritonX-100 of 0.2%, each 5 minutes; Reaction 37 DEG C × 30 minutes in dark wet box after all microslides of negative control group and positive controls are dry, PBS rinsing 3 times, each 5 minutes; Dark wet box is airtight lucifuge, the humidity box more than 80%.
9. all microslides after step 8. finally being processed add 50 ~ 100 microlitre diaminobenzidine (DAB) substrates, react 15 ~ 25 DEG C × 10 minutes;
10. the microslide haematoxylin after step 9. being processed or methyl green counterstain, use tap water after 5 seconds immediately; Gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting; Examine under a microscope result.
Step 1. described in distilled water be distilled water.
Step
described in microscope be that Germany produces Leica board DM4000B type microscope.
Step 9. described in diaminobenzidine (DAB) substrate be diaminobenzidine developer, add rear natural light basis of microscopic observation sperm.
For improving accuracy of detection, described pachymaran adopts following methods to extract: the grinding of 1000 grams, herbal raw material Poria cocos, cross 60 mesh sieves, be placed in 1000 milliliters, the physiological saline of 0.9% and soak 1 hour, the ratio of Poria cocos raw material and physiological saline is 1:1(weight ratio), be placed in the Chinese medicine extraction machine of 100 ~ 120 DEG C and add extraction heat 30 minutes, after taking out, filter and remove residue obtains Poria cocos extract, and the 1000 milliliters of 60 DEG C of dryings of Poria cocos extract are obtained rough pachymaran 320 grams in 2 hours; Take 100 grams of rough pachymarans to put in 500 milliliters of round-bottomed flasks, add sherwood oil 250 milliliters in 60 ~ 90 DEG C of backflows degreasing in 1 hour, filter, sherwood oil reclaims to obtain filter residue; Filter residue volatilizes after solvent with 80% ethanol, 250 milliliters of steeped overnight, 80 ~ 90 DEG C of refluxing extraction 2 times, each 1.5 hours, filter to obtain the dregs of a decoction, dregs of a decoction adding distil water 500 milliliters leaching got 40 minutes in 90 DEG C of warm lixiviates after 1 hour, filtered to obtain extract, again by extract 250 ml distilled water 90 DEG C temperature leaching 30 minutes, filter, merge 40 minutes gained are got in above-mentioned dregs of a decoction adding distil water 500 milliliters leaching in 90 DEG C of warm lixiviates filtrate after 1 hour, be evaporated to 75 milliliters and obtain concentrated filtrate; By concentrated filtrate deproteinization, add activated carbon decolorizing 2 times, suction filtration, filtrate adds 95% ethanol to be made to reach 80%, refrigerator hold over night containing amount of alcohol; Filter to obtain residue, in 60 DEG C of oven dry after residue washs with 95% ethanol, absolute ethyl alcohol, ether, acetone successively, obtain the pachymaran 210 grams of brown, the consumption of activated charcoal is that every 100 milliliters of concentrated filtrates add 1 gram of activated charcoal.
For improving testing result further, need above-mentioned pachymaran purifying.Select DEAE cellulose column to described pachymaran separation and purification: above DEAE cellulose column once: be packed into by pachymaran in DEAE cellulose column, afterwards, first take off liquid by the polysaccharide liquid that distilled water wash-out obtains a pachymaran component, then carry out gradient elution with 0.2 mol/L sodium hydroxid; Amount to upper DEAE cellulose column six times, each filling pachymaran 2.5 grams, collects the pachymaran eluent of whole six times, dialyses 3 days, must refine pachymaran 10.9 grams of white solids after vacuum freeze drying in flowing water.
The technology contents of the not detailed description of the present invention is known technology.
Claims (6)
1. detect a method for spermatoblast apoptosis and film integrality simultaneously, it is characterized in that: operation step is poly-as follows:
1. prepare hypotonic medium: in every 100 ml distilled waters, put into 735 milligrams of sodium citrates and 1350 milligrams of fructose make hypotonic medium, and make its ionic strength be 0.15(coefficient), osmotic pressure 150 rubs the solution of this mole;
2. to every 1 milliliter of step 1. described in hypotonic medium in add 0.1 milliliter of seminal fluid, mixing, in water temperature be in the water of 37 DEG C water-bath after 30 minutes seminal fluid after Hypotonic treatment, observe 200 sperms, the sperm ratio of calculating afterbody swelling;
3. get 1 milliliter of step 2. described in Hypotonic treatment after seminal fluid, with centrifugal treating under the centrifuge speed of 1000 revs/min 5 minutes, after stratification, abandon supernatant, gained seminal fluid sperm sediment phosphate buffer (PBS) is diluted to sperm concentration 1,000 ten thousand/milliliter, obtains dilution seminal fluid;
4. get step 3. described in dilution seminal fluid 0.2 milliliter and agar gel by volume 1:1 ratio mix, be placed in the centrifuge tube of 1.5 milliliters, hatch under room temperature 37 DEG C of conditions and must configure seminal fluid in 5 minutes;
5. aspiration step 4. described in configuration seminal fluid 50 microlitre insert on microslide, add a cover cover glass, under the condition of environment temperature 4 DEG C place 4 minutes; Described microslide is the microslide that surface is covered with the poly-D-lysine of thin layer;
6. the cover glass on microslide 5. described for step is removed, microslide to be immersed in pachymaran liquid 25 minutes, with the process that fixes to material on microslide; Pachymaran liquid forms by adding 1 gram of pachymaran configuration in every 10 ml physiological salines;
7. use phosphate buffer (PBS) embathe step 6. described fixing after microslide secondary, each 5 minutes; Then in the TritonX-100 of 10 milliliter 0.2%, embathing process 5 minutes by embathing rear microslide, embathing secondary with phosphate buffer more afterwards, each 5 minutes;
8. mixed liquor is prepared with 50 microlitre TdT and the fluorescein-labeled dUTP liquid mixing of 450 microlitres; 7. the several piece microslide after processing through step is divided into negative control group and positive controls, and the every block microslide wherein to negative control group adds the fluorescein-labeled dUTP liquid of 50 microlitre; Every block microslide of positive controls first adds 100 microlitre DNase1, reacts at 15 ~ 25 DEG C × 10 minutes, is processed by every block microslide afterwards, embathe 5 minutes, then embathe secondary with PBS in 10 milliliters in the TritonX-100 of 0.2%, each 5 minutes; Reaction 37 DEG C × 30 minutes in dark wet box after all microslides of negative control group and positive controls are dry, PBS rinsing 3 times, each 5 minutes; Dark wet box is airtight lucifuge, the humidity box more than 80%;
9. all microslides after step 8. finally being processed add 50 ~ 100 microlitre diaminobenzidine (DAB) substrates, react 15 ~ 25 DEG C × 10 minutes;
10. the microslide haematoxylin after step 9. being processed or methyl green counterstain, use tap water after 5 seconds immediately; Gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting; Examine under a microscope result.
2. a kind of method simultaneously detecting spermatoblast apoptosis and film integrality according to claim 1, is characterized in that: step 1. described in distilled water be distilled water.
3. a kind of method simultaneously detecting spermatoblast apoptosis and film integrality according to claim 1, is characterized in that: step
described in microscope be that Germany produces Leica board DM4000B type microscope.
4. a kind of method simultaneously detecting spermatoblast apoptosis and film integrality according to claim 1, it is characterized in that: step 9. described in diaminobenzidine (DAB) substrate be diaminobenzidine developer, add rear natural light basis of microscopic observation sperm.
5. a kind of method simultaneously detecting spermatoblast apoptosis and film integrality according to claim 1, it is characterized in that: the extracting method of described pachymaran is: the grinding of 1000 grams, herbal raw material Poria cocos, cross 60 mesh sieves, be placed in 1000 milliliters, the physiological saline of 0.9% and soak 1 hour, the ratio of Poria cocos raw material and physiological saline is 1:1(weight ratio), be placed in the Chinese medicine extraction machine of 100 ~ 120 DEG C and add extraction heat 30 minutes, after taking out, filter and remove residue obtains Poria cocos extract, the 1000 milliliters of 60 DEG C of dryings of Poria cocos extract are obtained rough pachymaran 320 grams in 2 hours, take 100 grams of rough pachymarans to put in 500 milliliters of round-bottomed flasks, add sherwood oil 250 milliliters in 60 ~ 90 DEG C of backflows degreasing in 1 hour, filter, sherwood oil reclaims to obtain filter residue, filter residue volatilizes after solvent with 80% ethanol, 250 milliliters of steeped overnight, 80 ~ 90 DEG C of refluxing extraction 2 times, each 1.5 hours, filter to obtain the dregs of a decoction, dregs of a decoction adding distil water 500 milliliters leaching got 40 minutes in 90 DEG C of warm lixiviates after 1 hour, filtered to obtain extract, again by extract 250 ml distilled water 90 DEG C temperature leaching 30 minutes, filter, merge 40 minutes gained are got in above-mentioned dregs of a decoction adding distil water 500 milliliters leaching in 90 DEG C of warm lixiviates filtrate after 1 hour, be evaporated to 75 milliliters and obtain concentrated filtrate, by concentrated filtrate deproteinization, add activated carbon decolorizing 2 times, suction filtration, filtrate adds 95% ethanol to be made to reach 80%, refrigerator hold over night containing amount of alcohol, filter to obtain residue, in 60 DEG C of oven dry after residue washs with 95% ethanol, absolute ethyl alcohol, ether, acetone successively, obtain the pachymaran 210 grams of brown, the consumption of activated charcoal is that every 100 milliliters of concentrated filtrates add 1 gram of activated charcoal.
6. a kind of method simultaneously detecting spermatoblast apoptosis and film integrality according to claim 5, it is characterized in that: with DEAE cellulose column to described pachymaran separation and purification: above DEAE cellulose column once: be packed into by pachymaran in DEAE cellulose column, afterwards, first take off liquid by the polysaccharide liquid that distilled water wash-out obtains a pachymaran component, then carry out gradient elution with 0.2 mol/L sodium hydroxid; Amount to upper DEAE cellulose column six times, each filling pachymaran 2.5 grams, collects the pachymaran eluent of whole six times, dialyses 3 days, must refine pachymaran 10.9 grams of white solids after vacuum freeze drying in flowing water.
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| CN107677648A (en) * | 2017-11-16 | 2018-02-09 | 南方医科大学南方医院 | The evaluation method of post operative colo-rectal cancer mesorectum sample integrality |
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