与骨骼相关的组织学实验技术方法 专区
来自 : 蚂蚁淘
EmbeddingBonesforFrozenSection:
- Fillanicebucketwithdryicepelletsandplacea500mlbeakerinthemiddle.
- Putapproximately200mlof2-MethylButaneinthebeakerandcoverthebucket.(TheMethylButanewillgetextremelycold,allowingyouto‘flashfreeze’yoursamples).
- LabelyourmoldsandfillthemoldswithFrozenEmbeddingMedium(ThermoShandon,Pittsburgh,PA).
- Immerseboneinembeddingmediumandadjustrightpositionasneeded.
- Test2-MethylButanewithdryicepellets,ifitdoesnotboil,itisready.
- Useaforcepstokeeptheembeddingmoldhorizontalyforfewsecondsuntiltheembeddingmediumisfrozeninpre-coldMethybutaneandallowittosittothebottomofthebeakerforatleast2minutestoensureitisfrozenallthewaythrough.
- Frozensamplescanbeplacedondryiceinthebucketuntilallsamplesaredone.Wrapyoursamplesinaluminiumfoil.
- Placeinasealedcontainerandstoreyoursamplesat-20�Cor-80�C.
TrapStain:
| BufferISolution: | | |
| SodiumAcetateAnhydrous(SigmaS-2889) | 9.2g | |
| L-(+)TartaricAcid(SigmaT-6521) | 11.4g | |
| DistilledWater | 950ml | |
| GlacialAceticAcid | 2.8ml | |
- DissolveandadjustpHto4.7-5.0with5MSodiumHydroxide
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|
- (5MNaOH:SodiumHydroxidePellets50g,DistilledWater—250ml)
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| BufferIISolution: | | |
| NaphtholAS-BIPhosphate(SigmaN-2125)storeat–20� | 0.1g | |
| EthyleneGlycolMonoethylEther(SigmaE-2632) | 5ml | |
| BufferIIISolution: | | |
| SodiumNitrite(SigmaS-2252) | 1g | |
| DistilledWater | 20ml | |
| BufferIVSolution: | | |
| PararosanilineChloride(SigmaP-3750) | 1g | |
| 2NHCL | 20ml | |
|
|
- (2NHCL:HCL,83ml;Distilledwater,417ml)
|
StainingProcedure:
- Preheatto37°C2Coplinjarswith50mlofBufferISolutioneach.
- TakeoneCoplinjarandadd0.5mlofBufferIISolution,addslidesandincubateat37°Cfor45minus.
- Afewminbeforethetimeisup,mix1mlofBufferIIISolutionand1mlofBufferIVsolutionfor30secandletitsitfor2min.
- AddthismixedsolutiontotheotherCoplinjarof50mlBufferISolution,mixandaddtheslideswithoutrinsing.
- Incubateatroomtemp~5min.
- Rinse,counterstainwithhematoxylinfor40secandputslidesin0.05%Ammoniawater.
- Dehydratethroughalcohol,clearinxyleneandmount.
AlizarinRedSStaining-Calcium |
| | | |
AlizarinRedSSolution: | | |
AlizarinRedS | 2gm | |
DistilledWater | 100ml | |
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|
|
Acetone–Xylene: | | |
Acetone | 25ml | |
Xylene | 25ml | |
Makefresh. |
Procedure:
- Rinseslidesrapidlywithdistilledwater.
- AlizarinredSsolution,30secto3min,checkingmicroscopicallyfortheorange-redcolor.
- Shakeoffexcessdye.
- Acetone:-20dips.
- Acetone-xylene:-20dips.
- Clearinxylene,mountinpermount.
GoldnerTrichromeStaining |
| | | |
WeigertHematoxyline: | | |
| | | |
| SolutionA | | |
| Hematoxyline | 5g | |
| 95%Alcohol | 500ml | |
| | | |
| SolutionB | 5g | |
| Ferricchloride | 5.8gl | |
| Deionizedwater | 500ml | |
| ConcentratedChlorhydricAcide | 5.0ml | |
- 24hoursbeforeuse,mixsolutionsAandBinequalamountsStablefor8days.
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|
| Fushine-Ponceau: | | |
| Fushineacid | 0.167g | |
| PonceauS | 0.667g | |
| Deionizedwater | 500ml | |
| AceticAcid(glacial) | 1.0ml | |
|
|
| OrangeG: | | |
| Phospho-molyBDicAcid | 25g | |
| Deionizedwater(Shakewell) | 500ml | |
| AddOrangeG | 10g | |
|
|
| LightGreen: | | |
| Lightgreen | 1.5g | |
| Deionizedwater | 500ml | |
| AceticAcid(glacial) | 1ml | |
|
| AceticAcid1% | | |
StainingProcedure:
- Putslidesindeionizedwater.
- MordantinBouin’sFixative(PolyScientificInc.Catalog#s129-16oz)atroomtemperatureovernight.
- Rinsewithdeionizedwaterfrequentlyfor10minutes.
- WeightHematoxyline15minutes.
- Rinsewithdeionizedwaterfrequentlyfor10minutes.
- Fushine-ponceau30minutes.
- Rinsewith1%aceticacidrapidly.
- OrangeG8minutes.
- Rinsewith1%aceticacidrapidly.
- LightGreen20minutes
- Rinsewith1%aceticacidrapidly.
- Dehydratethroughalcohol,clearinxyleneandmount.
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