请使用支持JavaScript的浏览器! 与骨骼相关的组织学实验技术方法 专区 _蚂蚁淘,【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
当前位置: > 首页 > 技术文章 >
与骨骼相关的组织学实验技术方法 专区
来自 : 蚂蚁淘

EmbeddingBonesforFrozenSection:

    1. Fillanicebucketwithdryicepelletsandplacea500mlbeakerinthemiddle.
    2. Putapproximately200mlof2-MethylButaneinthebeakerandcoverthebucket.(TheMethylButanewillgetextremelycold,allowingyouto‘flashfreeze’yoursamples).
    3. LabelyourmoldsandfillthemoldswithFrozenEmbeddingMedium(ThermoShandon,Pittsburgh,PA).
    4. Immerseboneinembeddingmediumandadjustrightpositionasneeded.
    5. Test2-MethylButanewithdryicepellets,ifitdoesnotboil,itisready.
    6. Useaforcepstokeeptheembeddingmoldhorizontalyforfewsecondsuntiltheembeddingmediumisfrozeninpre-coldMethybutaneandallowittosittothebottomofthebeakerforatleast2minutestoensureitisfrozenallthewaythrough.
    7. Frozensamplescanbeplacedondryiceinthebucketuntilallsamplesaredone.Wrapyoursamplesinaluminiumfoil.
    8. 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
  • Bringtotalvolumeto1L
  • (5MNaOH:SodiumHydroxidePellets50g,DistilledWater—250ml)
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
  • Heatto60�;Cfor5mindonotboil.
  • Filterthroughkimwipe.
  • (2NHCL:HCL,83ml;Distilledwater,417ml)

StainingProcedure:

  1. Preheatto37°C2Coplinjarswith50mlofBufferISolutioneach.
  2. TakeoneCoplinjarandadd0.5mlofBufferIISolution,addslidesandincubateat37°Cfor45minus.
  3. Afewminbeforethetimeisup,mix1mlofBufferIIISolutionand1mlofBufferIVsolutionfor30secandletitsitfor2min.
  4. AddthismixedsolutiontotheotherCoplinjarof50mlBufferISolution,mixandaddtheslideswithoutrinsing.
  5. Incubateatroomtemp~5min.
  6. Rinse,counterstainwithhematoxylinfor40secandputslidesin0.05%Ammoniawater.
  7. Dehydratethroughalcohol,clearinxyleneandmount.

    AlizarinRedSStaining-Calcium
    AlizarinRedSSolution:
    AlizarinRedS
    2gm
    DistilledWater
    100ml
    • MixthesolutionandadjustthepHto4.1-4.3using0.5%ammoniumhydroxide.ThepHiscritical-makefresh.
    Acetone–Xylene:
    Acetone
    25ml
    Xylene
    25ml
    Makefresh.

    Procedure:

    1. Rinseslidesrapidlywithdistilledwater.
    2. AlizarinredSsolution,30secto3min,checkingmicroscopicallyfortheorange-redcolor.
    3. Shakeoffexcessdye.
    4. Acetone:-20dips.
    5. Acetone-xylene:-20dips.
    6. Clearinxylene,mountinpermount.

    GoldnerTrichromeStaining
    WeigertHematoxyline:
    SolutionA
    Hematoxyline
    5g
    95%Alcohol
    500ml
    SolutionB
    5g
    Ferricchloride
    5.8gl
    Deionizedwater
    500ml
    ConcentratedChlorhydricAcide
    5.0ml
    • 24hoursbeforeuse,mixsolutionsAandBinequalamountsStablefor8days.
    Fushine-Ponceau:
    Fushineacid
    0.167g
    PonceauS
    0.667g
    Deionizedwater
    500ml
    AceticAcid(glacial)
    1.0ml
    • Shakewellandfilter.
    OrangeG:
    Phospho-molyBDicAcid
    25g
    Deionizedwater(Shakewell)
    500ml
    AddOrangeG
    10g
    • Shakewellandfilter.
    LightGreen:
    Lightgreen
    1.5g
    Deionizedwater
    500ml
    AceticAcid(glacial)
    1ml
    • Shakewellandfilter.
    AceticAcid1%

    StainingProcedure:

      1. Putslidesindeionizedwater.
      2. MordantinBouin’sFixative(PolyScientificInc.Catalog#s129-16oz)atroomtemperatureovernight.
      3. Rinsewithdeionizedwaterfrequentlyfor10minutes.
      4. WeightHematoxyline15minutes.
      5. Rinsewithdeionizedwaterfrequentlyfor10minutes.
      6. Fushine-ponceau30minutes.
      7. Rinsewith1%aceticacidrapidly.
      8. OrangeG8minutes.
      9. Rinsewith1%aceticacidrapidly.
      10. LightGreen20minutes
      11. Rinsewith1%aceticacidrapidly.
      12. Dehydratethroughalcohol,clearinxyleneandmount.

免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。
相关文章