CN103776679B - A kind of method for reducing biological sample background fluorescence - Google Patents
A kind of method for reducing biological sample background fluorescence Download PDFInfo
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- CN103776679B CN103776679B CN201410043251.8A CN201410043251A CN103776679B CN 103776679 B CN103776679 B CN 103776679B CN 201410043251 A CN201410043251 A CN 201410043251A CN 103776679 B CN103776679 B CN 103776679B
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Abstract
The invention discloses a kind of method for reducing biological sample background fluorescence, belongs to technical field of bioengineering.The method as light absorber, is enabled Quick uniform using high concentration ethanol solution dissolving sudan black B stain liquid and is penetrated in biological tissue using Sudan black B.Substantially reduced using the method gained biological sample background fluorescence, improve the signal contrast of fluorescence imaging.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly to a kind of method for reducing biological sample background fluorescence.
Background technology
Epi-fluorescence micro-imaging is one of most-often used microtechnic in biological study, itself and fluorescin（GFP and
Its derivative, XFPs）, immunofluorescence and fluorescence probe mark method combine after, zoology, botany, microbiology,
The fields such as immunology, pathology and pharmacy are widely used.
However, fall to penetrating formula fluorescence imaging there is a problem of one very serious, i.e. the interference of background fluorescence, especially to thicker
Biological tissue's block.The not only possible useful signal for causing mistake of background fluorescence, even more so that the signal on focal plane is annihilated.And
The fine structure of local in biological study, is usually not only needed, and is chased after with greater need for large-scale structure being carried out for thick tissue block
Track.The research of such as brain 26S Proteasome Structure and Function, it is often necessary to which the Mouse brain of millimeter even cm size is imaged.Therefore,
The problems demand of this large scale sample background fluorescence is solved.
Disturb for background fluorescence, there are several settling modes at present：One kind is outer glimmering from imaging technique angle elimination focal plane
The impact of optical signal.Common settling mode is the technology that is cut into slices using light, and such as confocal laser scanning microscope, CLSM, multi-photon shows
Micro mirror, mating plate illumination and Structured Illumination microscope.But these modes all have some limitations.Such as confocal laser
Flying-spot microscope and multiphoton microscope adopt spot scan mode, and image taking speed is slow, visual field is little, to needing during large scale imaging samples
Consume a longer time.Mating plate illumination microscope is limited to the operating distance of object lens, it is impossible to carried out into using the object lens of high na value
Picture, causes imaging resolution not high.Structured Illumination microscope is sensitive to noise such as shot noise, shot after decoding algorithm
Noise is greatly increased, it is difficult to be applied to the stronger sample of thicker, background fluorescence.Another kind is glimmering using chemical means reduction background
Light.Such as describe to reduce unwanted light in cell solution using multiple dyestuffs in patent U.S.Pat.No.6,221,612
The method of transmitting.For background fluorescence caused by autofluorescence, also there is researcher to attempt being gone with various coloring agents always
Remove, such as document " Reduction of lipofuscin-like autofluorescence in fluorescently
Labeled tissue. (Schnell, S.A., W.A.Staines and M.W.Wessendorf, Journal of
Histochemistry&Cytochemistry47(1999)719-730）”、“Working with GFP in the brain.
(Doyle,K.P.,R.P.Simon,A.Snyder,et al.,Biotechniques 34(2003)492-494)”、“Sudan
Black B treatment reduces autofluorescence and improves resolution of in situ
hybridization specific fluorescent signals of brain sections.(Oliveira,V.C.,
R.C.V.Carrara,D.L.C.Simoes,et al.,Histology and Histopathology25,(2010)1017-
1024”、“Sudan Black B Reduces Autofluorescence in Murine Renal Tissue.(Sun,Y.,
H.Yu,D.Zheng,et al.,Archives of Pathology&Laboratory Medicine135(2011)1335-
1342)”.However, current colouring method is all confined in the section of cell solution or tens microns, to grade even centimetre
Biological tissue's block of level is not studied.And when sample size increases, frequently result in that dyeing time is slow, it is uneven, real to dye
Test the phenomenons such as flow process is loaded down with trivial details.Such as during sulphur a beautiful gem dyeing Mouse Whole Brain dyeing color separation is generally needed access to one month, Golgi-Cox methods
Dyeing Mouse Whole Brain even needs half a year.Therefore, a kind of suitable light absorber for being applied to specific fluorescent material is selected, is found
Its suitable use condition so as to quickly and evenly penetrate in biological tissue, may be also required to substantial amounts of research.
Content of the invention
The purpose of the present invention is provided one kind and substantially reduces biological sample fluorescence background for solving the above problems, and is improved
The method of fluorescence imaging signal contrast.
The technical solution adopted in the present invention is：
A kind of method for reducing biological sample background fluorescence, comprises the following steps：
（1）The paraformaldehyde solution that the mass percent that biological sample frozen water is precooled is 4% is fixed 6～24h；
（2）By step（1）The concentration that the biological sample frozen water for obtaining is precooled is that the PBS rinsing liquids of 0.01mol/L are clear
3～24h is washed, therebetween replacing Fresh rinse solution 3～5 times；
（3）By step（2）It is 50% and 70% that the biological sample for obtaining is sequentially placed into the mass percent of frozen water precooling
Serial dehydration, each 15min～2h is carried out in ethanol solution；
（4）By step（3）The biological sample for obtaining is put into dyeing 30min～48h in sudan black B stain liquid；
（5）By step（4）The concentration that the biological sample frozen water that obtains is precooled be 0.01mol/L PBS rinsing liquids 6～
24h, changes Fresh rinse solution 3～5 times therebetween.
Preferably, the step（1）The size of middle biological sample is less than 2 centimeter squares.
Further, the step（4）Middle sudan black B stain liquid is dense by the ethanol dissolving of mass percent 90～100%
Spend the sudan black B stain liquid for 0.5～5w/w ‰ (g/kg) to prepare.
Further, the step（4）Depending on middle sudan black B stain liquid dyeing time is according to the size of biological sample, 1 milli
Block staining's time within meter Hou Du is 30min～2h, and block staining's time of 1～5 mm of thickness is 1～12 little
When, block staining's time of 5 millimeters～2 cm thicks is 12～48h.
Preferably, the biological sample can be the animal tissue of any transmitting fluorescence.
The present invention has advantages below：
The present invention is that a kind of light absorber is dispersed in biological sample, the absorption spectrum of the light absorber with described
Exciting or emission spectrum overlap for biological sample background fluorescence, using Sudan black B as light absorber, molten using high concentration ethanol
Liquid dissolving sudan black B stain liquid enables Quick uniform and penetrates in biological tissue.The method gained biological sample background is glimmering
Light is substantially reduced, and improves the signal contrast of fluorescence imaging.The inventive method is with low cost, and operating procedure is simple, during process
Between short, be suitable for promoting the use of in common laboratory.The method of the present invention is can apply in cm size biological sample simultaneously, is processed
Time is short.
Description of the drawings
Fig. 1 is that the embodiment of the present invention 1 is micro- using the background fluorescence contrast before and after sudan black B stain liquid process biological specimen
Mirror figure.
Specific embodiment
The present invention will be further described in detail with embodiment below in conjunction with the accompanying drawings.
Embodiment 1
A kind of method for reducing biological sample background fluorescence is present embodiments provided, is comprised the following steps：
Brain block of the biological specimen for 3 mm of thickness of Thy1-eYFP-H mouse, imaging system are that Olympus IX71 wide fields are glimmering
Light microscope.
（1）After Thy1-eYFP-H line transgenic mices are anaesthetized with 1% yellow Jackets, filled by heart left ventricle
The PBS solution of the 0.01mol/L that 37 DEG C of note 3 minutes, after blood is rinsed well, the quality percentage that perfusion frozen water is precooled immediately
Than 4% paraformaldehyde fixer, and continue 1 hour.Then, Mouse Whole Brain is taken out, is cut into the brain block of 3 mm of thickness, is placed again into
12 hours are fixed after in the 4% paraformaldehyde fixer that frozen water is precooled.2.5% sucrose, solvent is added for concentration to be in fixer
The PBS of 0.01mol/L.
（2）After fixation terminates, full brain sample is put in PBS of the concentration of frozen water precooling for 0.01mol/L and rinses 12
Hour, more renew liquid 3 times therebetween, with the paraformaldehyde fixer that thoroughly cleaning falls remnants.
（3）Then, by brain block sample be sequentially placed into frozen water precooling concentration be 50% and 70% ethanol solution in carry out ladder
Degree dehydration, each 30min.
（4）After dehydration terminates, full brain sample is put into 0.5 ‰ sudan black B stains of the 95% ethanol preparation of frozen water precooling
In liquid, dye 6 hours in the case of lucifuge.Sudan black B stain liquid is filtered before sample is processed.
（5）Finally, the concentration that the dyeing brain block of 6 hours is placed into frozen water precooling is rinsing 12 in the PBS of 0.01mol/L
Hour, more renew liquid 3 times therebetween, with the ethanol solution that thoroughly cleaning falls remnants.With reference to Fig. 1, a is the sample before dyeing, is imaged
120 milliseconds of time for exposure；B be dyeing after sample, 300 milliseconds of Imagewise exposure time., it is apparent that passing through from Fig. 1
The method of the present embodiment, biological sample background fluorescence are substantially reduced, and improve the signal contrast of fluorescence imaging.
Embodiment 2
A kind of method for reducing biological sample background fluorescence is present embodiments provided, is comprised the following steps：
Biological specimen is Thy1-eYFP-H Mouse Whole Brain samples.
（1）After Thy1-eYFP-H line transgenic mices are anaesthetized with 1% yellow Jackets, filled by heart left ventricle
The PBS solution of the 0.01mol/L that 37 DEG C of note 3 minutes, after blood is rinsed well, the quality percentage that perfusion frozen water is precooled immediately
Than 4% paraformaldehyde fixer, and continue 1 hour.Then, Mouse Whole Brain is taken out, is placed again into 4% poly first of frozen water precooling
24 hours are fixed after in aldehyde fixer.2.5% sucrose is added in fixer, and solvent is the PBS that concentration is 0.01mol/L.
（2）After fixation terminates, full brain sample is put in PBS of the concentration of frozen water precooling for 0.01mol/L and rinses 24
Hour, more renew liquid 5 times therebetween, with the paraformaldehyde fixer that thoroughly cleaning falls remnants.
（3）Then, by full brain sample be sequentially placed into frozen water precooling concentration be 50% and 70% ethanol solution in carry out ladder
Degree dehydration, 2 hours every time.
（4）After dehydration terminates, full brain sample is put into 3 ‰ sudan black B stain liquid of the 90% ethanol preparation of frozen water precooling
In, dye 48 hours in the case of lucifuge.Sudan black B stain liquid is filtered before sample is processed.
（5）Finally, the concentration that the dyeing mouse brain of 48 hours is placed into frozen water precooling is rinsing in the PBS of 0.01mol/L
24 hours, more renew liquid 4 times therebetween, with the ethanol solution that thoroughly cleaning falls remnants.
Embodiment 3
A kind of method for reducing biological sample background fluorescence is present embodiments provided, is comprised the following steps：
Brain block of the biological specimen for 1 mm of thickness of Thy1-eYFP-H mouse.
（1）After Thy1-eYFP-H line transgenic mices are anaesthetized with 1% yellow Jackets, filled by heart left ventricle
The PBS solution of the 0.01mol/L that 37 DEG C of note 3 minutes, after blood is rinsed well, the quality percentage that perfusion frozen water is precooled immediately
Than 4% paraformaldehyde fixer, and continue 1 hour.Then, Mouse Whole Brain is taken out, is cut into the brain block of 1 mm of thickness, is placed again into
6 hours are fixed after in the 4% paraformaldehyde fixer that frozen water is precooled.2.5% sucrose, solvent is added for concentration to be in fixer
The PBS of 0.01mol/L.
（2）After fixation terminates, full brain sample is put into rinsing 3 in PBS of the concentration of frozen water precooling for 0.01mol/L little
When, more renew liquid 4 times therebetween, with the paraformaldehyde fixer that thoroughly cleaning falls remnants.
（3）Then, by full brain sample be sequentially placed into frozen water precooling concentration be 50% and 70% ethanol solution in carry out ladder
Degree dehydration, 15 minutes every time.
（4）After dehydration terminates, full brain sample is put into 5 ‰ sudan black B stains of the 100% ethanol preparation of frozen water precooling
In liquid, dye 30 minutes in the case of lucifuge.Sudan black B stain liquid is filtered before sample is processed.
（5）Finally, the concentration that the dyeing mouse brain of 30 minutes is placed into frozen water precooling is rinsing in the PBS of 0.01mol/L
15 hours, more renew liquid 3 times therebetween, with the ethanol solution that thoroughly cleaning falls remnants.
The present invention is that a kind of light absorber is dispersed in biological sample, the absorption spectrum of the light absorber with described
Exciting or emission spectrum overlap for biological sample background fluorescence, using Sudan black B as light absorber, molten using high concentration ethanol
Liquid dissolving sudan black B stain liquid enables Quick uniform and penetrates in biological tissue.The method gained biological sample background is glimmering
Light is substantially reduced, and improves the signal contrast of fluorescence imaging.The inventive method is with low cost, and operating procedure is simple, during process
Between short, be suitable for promoting the use of in common laboratory.The method of the present invention is can apply in cm size biological sample simultaneously, is processed
Time is short.
It should be noted last that, above example is only unrestricted in order to technical scheme to be described, although ginseng
The present invention is described in detail according to preferred embodiment, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent, without deviating from the spirit and scope of technical solution of the present invention, which all should be covered
In the middle of scope of the presently claimed invention.
Claims (4)
1. a kind of reduce biological sample background fluorescence method, it is characterised in that comprise the following steps：
(1) paraformaldehyde solution that the mass percent that biological sample frozen water is precooled is 4% is fixed 6～24h, fixer
2.5% sucrose of middle addition；
(2) biological sample that step (1) the is obtained PBS rinsing liquids that the concentration that frozen water is precooled is 0.01mol/L clean 3～
24h, changes Fresh rinse solution 3～5 times therebetween；
(3) by the biological sample that step (2) is obtained be sequentially placed into frozen water precooling mass percent be 50% and 70% second
Serial dehydration, each 15min～2h is carried out in alcoholic solution；
(4) biological sample that step (3) is obtained is put into dyeing 30min～48h in sudan black B stain liquid；The Sudan black B dye
Color liquid is prepared into by the sudan black B stain liquid that the ethanol concentration of ordinary dissolution of mass percent 90～100% is 0.5～5w/w ‰
Arrive；
(5) concentration that the biological sample frozen water for obtaining step (4) is precooled is the PBS 6～24h of rinsing liquid of 0.01mol/L,
Fresh rinse solution 3～5 time is changed therebetween.
2. according to claim 1 reduce biological sample background fluorescence method, it is characterised in that in step (1)
The size of biological sample is less than 2 centimeter squares.
3. according to claim 1 reduce biological sample background fluorescence method, it is characterised in that in step (4)
Depending on sudan black B stain liquid dyeing time is according to the size of biological sample, the block staining within 1 mm of thickness is at the time
30min～2h, block staining's time of 1～5 mm of thickness is 1～12 hour, and the tissue block of 5 millimeters～2 cm thicks contaminates
The color time is 12～48h.
4. according to any one of claims 1 to 3 minimizing biological sample background fluorescence method, it is characterised in that described
Biological sample can be the animal tissue of any transmitting fluorescence.
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