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Histology 組織學相關產品
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   一點閃爍計數液



      Edith
      2012.02.04
切片
冷凍切片 frozen sections
 急速冷凍▸切片▸染色
 固定▸脫水▸包埋▸切片▸染色

石蠟切片 paraffin-embedded sections
 固定▸脫鈣▸脫水▸澄清▸浸潤▸包埋▸切片▸染色
固定 Fixation
保持細胞、組織的固有形態和結構
使原來的軟組織硬化,而使得隨後的操作程序
容易進行

常使用   福馬林相關溶液
ex. 中性福馬林 (10% Buffered neutral formalin)
   溴化氨福馬林溶液 (Formalin Ammonium
   Bromide Solution ; FAB)
   福馬林-酒精-醋酸溶液 (Formalin-
   Alcohol-Acetic Acid Solution; FAA)
Mirsky's Fixative
                               HS-101 & HS-102
冷凍切片 / 石蠟切片
Saponin( 皂素 )–Glyoxal( 乙二醛 ) type fixative
相較於 formalin:
 o reduced toxicity
 o superior preservation of epitopes for
    IHC & ICC
 o improved retention of tissue morphology
 x tissue blocks < 3mm
脫鈣 Decalcification
骨組織及鈣化病灶,經過固定後,必先將鈣鹽
除去使組織軟化,才能進行常規切片。
如脫鈣不全則切片易撕開或碎裂並損傷切片刀刃。
Mineral Acids 礦物酸 / 無機酸 (ex. Nitric Acid, Hydrochloric Acid)
               作用快 ; 適用於質密的骨 sample
                 Damage Cellular Morphology
Organic Acids 有機酸 (ex. Acetic Acid, Formic Acid)
               作用較慢 ; 適用於 bone marrow, other soft tissues
EDTA
            作用最慢
          不適合常規使用

               增加脫鈣速度則犧牲細胞微細構造
HS-104
Calci-Clear
                                  HS-105
Calci-Clear Rapid
Moderate speed decalcifier.
▸   Mildness balanced with performance
▸ Minimized loss of tissue structure
▸ Excellent staining characteristics




                增加脫鈣速度則犧牲細胞微細構造
脫水 Dehydration
澄清 Clearing
將已固定組織內之游離水份脫除,便於無法與脫水
劑 , 水互溶之包埋劑浸潤
乙醇
 HS-300
 (95% Ethanol/5% Methanol)

Xylene 二甲苯
 Histo-Clear
 Histo-Clear II
HS-200
Histo-Clear
                HS-202
Histo-Clear II
無毒 + Biodegradable
 使用 Histo-Clear 的客戶較多
 成份略不同
 Histo-Clear II 較無侵蝕性 , 但用以替換 xylene 可能需
 較長的 incubation time
 Histo-Clear II 需與特定的 mounting medium 搭配使用 ,
 ex. Omnimount(HS-106)
                                                   HS-100
                                   Histosol
                          Flash point 較 xylene 高
浸潤 Infiltration
包埋 Embedding
 包埋劑呈液態時可經由置換作用
 而進入組織間隙 , 爾後凝固以便
 切片
 常用之包埋劑 :
     石蠟 (Paraffin)
    賽勞定 (Celloidin)
切片 Sectioning
石
臘
旋
轉               15-25μm
式                 ↓
切               3-5μm
片
機
染色 Staining


Antigen Retrieval
Protocol 裡的
xylene 亦可用
Histo-Clear 取代 !
染劑 Stains
             HS-504
Alcian Blue
acid mucopolysaccharides 酸性粘多醣體 ; mast cell differentiation
               HS-518
Basic Fuchsin 鹼性品紅 / 鹼性復紅 / 鹼性洋紅
Main ingredient of Schiff's reagent, a pH indicator, and stain for
glycoproteins and mucopolysaccharide proteins.
               HS-506
Biebrich Scarlet 猩紅質
Plasma stain substituted for Acid Fuchsin in Masson's trichrome.
                  HS-516
Fast Green FCF
Fibre stain substituted for Light Green SF Yellowish in Masson's
                                                      trichrome.
染劑 Stains
                        HS-603
Bromophenol Blue
Commonly used tracking dye in electrophoresis.
               HS-601
Amido Black
法醫學染色:強化微弱血跡
            HS-606
Methyl Green 甲 綠
               基
In conjunction with Pyronin Y will differentiate RNA and DNA.
            HS-607
Pyronin Y                                #61

In conjunction with Methyl Green will differentiate RNA and DNA.
                                  HS-525
                     Methylene Blue 亞 藍
                                     甲
                     A sensitive stain specific for DNA and RNA.
封片 Mounting
封片 Mounting
封片膠 / 封蓋劑 ex. 阿拉伯膠、加拿大膠 ...
Mounting Media ─ aqueous
               ─ non-aqueous/organic

先將滴一滴封片膠在組織中央,已 45 度角緩緩將蓋
玻片蓋上,再以鑷子輕壓將氣泡趕出
HS-103
Histomount
Classic choice in synthetic mounting media
                   HS-106
Hydromount
Nonfluorescing aqueous mounting medium
Effective for frozen sections, amyloid, and immunofluorescent staining
procedures
May be removed by soaking the slides in warm saline
                  HS-110
Omnimount
Universally compatible synthetic mounting medium. Ideally suited for
use with HistoClear II.
Higher flash point and Lower toxicity than xylene based mountants
Provides both a safer work environment and reduces shipping costs
順便一併提一下 ...          Anti-Fade
* 不是所有 Mounting Solution 都含有 Anti-Fade!*
      ReadiUse™ Microscope Mounting Solution ( 編號 20009)
      FluoroQuest™ Anti-fading Kit I *for Slide* ( 編號 20001)
      FluoroQuest™ Anti-fading Kit II *for Plate* ( 編號 20003)
                   AAT : 20009 不含 Anti-Fade 成份
      Fluoromount( 編號 NB900-66726)( 含 Anti-Fade)
      We do not add anti-fading compounds to our media in order to allow
      the maximum flexibility in their use, but they are compatible with
      all the common anti-fade additives- your customer can add, for
      example, DABCO to Hydromount, and achieve excellent result.
      i-BRITE Plus( 編號 SF40000-10)( 含 Anti-Fade)
      Mounting Medium, Aqueous, BioAssay™ ( 編號 M4679-01)
      Mounting Medium, Organic, BioAssay™ ( 編號 M4679)
  USB : There is no antifade added to either M4679 or M4679-01.
參考連結
                      Tissue Preparation for Light Microscopy
                      http://www.youtube.com/watch?v=TLm37BbR1mo


Protocols Online | Archive | Histology
http://protocolsonline.com/category/histology/

獸醫科技資訊網:參考手冊  動物傳染病實驗室檢驗方法  總論                                   第 二  
                                                                     十 章
病 學 查   http://vettech.nvri.gov.tw/Articles/handbook/916.html
 理 檢 法

                               Rotary Microtome Section
                 http://www.youtube.com/watch?v=KnMdSgd5mts
                                   Immunohistochemistry
                 http://www.youtube.com/watch?v=mpK5ojrXQs0
液相閃爍計數 LSC
Liquid Scintillation Counting
閃爍計數器為利用某些物質所產生的閃爍光來偵
測游離輻射
其檢體測試形式可分為液體或固體
液體閃爍計數器是利用閃爍材料如螢光劑將放射
性同位素之輻射能轉變為光能再由光電管將光
能轉為電脈衝進而測定輻射線的數量及能量
      成大 - 藥理學研究所 - 公用儀器 -B- 計數器說明
                 http://www.ncku.edu.tw/~pharma/v2011/8-2.php
液相閃爍計數 LSC
Liquid Scintillation Counting
          Cocktails ─
            1.Solvent
                  溶解度
                  collector & conductor of energy
                  must not quench Scintillator
            2.Scintillator
              -   primary
                   能被 excited solvent molecules 激發
              -   secondary
                   wavelength shifters( 早期 )
                   improve efficiency( 現在 )
液相閃爍計數 LSC
Liquid Scintillation Counting
液相閃爍計數 LSC
Liquid Scintillation Counting
- Metabolic studies
- Protein and DNA analysis studies

                細胞增生實驗
 (3H-Thymidine Incorporation/Cell Proliferation Assay)
利用活細胞合成 DNA 的過程,使其攝入放射性 3H-Thymidine ,
             依其放射強度代表細胞數目多寡
一排一排地將細胞 收集 (Cell Harvester) 至濾紙 ( 玻璃纖維 ) 上,
然後將含有 DNA 的濾紙剪下,置入計測瓶內,每個計測瓶加 1.5 - 2
      ml 閃爍計數液,置入 Beta counter 內計測
液相閃爍計數 LSC
Liquid Scintillation Counting


細胞收集器
(Cell Harvester)
液相閃爍計數 LSC
 Liquid Scintillation Counting
液態閃爍計數器
(Liquid Scintillation Analyzer, β-Counter)
 國立陽明大學
 高雄醫學大學
   臺灣大學
 國衛院育成中心 中興大學
   東海大學 中山醫學大學
        中臺科技大學
        屏東科技大學
          ...
液相閃爍計數 LSC
Liquid Scintillation Counting
Scintillation Vials
  Precision Molded
  Highest Quality HDPE
  Leak-Tight Seal
  Economical
                                        SVC-20
                                20 ml
                                       SVC-08
                                8 ml
                                       SVC-06
                                6 ml
閃爍計數液 LSC Cocktails
The Waste Disposal Issues
      in Scintillation Counting
   The components of the LSC cocktail may represent a hazard or a
 disposal problem in addition to the radioactivity.
   For many experiments, only a small percentage of the samples
 counted will have significant radioactivity, so disposal of the LSC
 is the predominating issue.
   Fortunately, biodegradable LSC cocktails have been developed,
 such as N.D.s' Ecoscint fluids and Uniscint BD, which
 substantially reduce the difficulty of disposing of LSC waste.
閃爍計數液 LSC Cocktails
                           LS-273
 Ecoscint A
 The Ideal Universal Scintillation Cocktail
 All Around Performance
 High Efficiency with High Sample Holding
 Biodegradable, Reduced Toxicity Solvent
 Biodegradable
 High Flash Point (>150°C)
 Exce ent for:
     ll
  Agar e gels, aqueous sa
      os                 mples, biologica samples, environmental samples, glass
                                         l
  fibe or paper filters plasma, polyacry mide gels, salt solutions, sucrose,
      r                ,                 la
  solu ized tissues.
      bl
                    Useful for :
                      Acidic sam es, alkaline sam es, catecholamine assays, high
                                pl               pl
                      salt solut ns, tissue homog ates.
                                io               en
Fin.

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Histology Products and Techniques Guide

  • 1. Histology 組織學相關產品 + 一點閃爍計數液 Edith 2012.02.04
  • 2. 切片 冷凍切片 frozen sections 急速冷凍▸切片▸染色 固定▸脫水▸包埋▸切片▸染色 石蠟切片 paraffin-embedded sections 固定▸脫鈣▸脫水▸澄清▸浸潤▸包埋▸切片▸染色
  • 3. 固定 Fixation 保持細胞、組織的固有形態和結構 使原來的軟組織硬化,而使得隨後的操作程序 容易進行 常使用 福馬林相關溶液 ex. 中性福馬林 (10% Buffered neutral formalin) 溴化氨福馬林溶液 (Formalin Ammonium Bromide Solution ; FAB) 福馬林-酒精-醋酸溶液 (Formalin- Alcohol-Acetic Acid Solution; FAA)
  • 4.
  • 5. Mirsky's Fixative HS-101 & HS-102 冷凍切片 / 石蠟切片 Saponin( 皂素 )–Glyoxal( 乙二醛 ) type fixative 相較於 formalin: o reduced toxicity o superior preservation of epitopes for IHC & ICC o improved retention of tissue morphology x tissue blocks < 3mm
  • 6. 脫鈣 Decalcification 骨組織及鈣化病灶,經過固定後,必先將鈣鹽 除去使組織軟化,才能進行常規切片。 如脫鈣不全則切片易撕開或碎裂並損傷切片刀刃。 Mineral Acids 礦物酸 / 無機酸 (ex. Nitric Acid, Hydrochloric Acid) 作用快 ; 適用於質密的骨 sample Damage Cellular Morphology Organic Acids 有機酸 (ex. Acetic Acid, Formic Acid) 作用較慢 ; 適用於 bone marrow, other soft tissues EDTA 作用最慢 不適合常規使用 增加脫鈣速度則犧牲細胞微細構造
  • 7. HS-104 Calci-Clear HS-105 Calci-Clear Rapid Moderate speed decalcifier. ▸ Mildness balanced with performance ▸ Minimized loss of tissue structure ▸ Excellent staining characteristics 增加脫鈣速度則犧牲細胞微細構造
  • 8. 脫水 Dehydration 澄清 Clearing 將已固定組織內之游離水份脫除,便於無法與脫水 劑 , 水互溶之包埋劑浸潤 乙醇 HS-300 (95% Ethanol/5% Methanol) Xylene 二甲苯 Histo-Clear Histo-Clear II
  • 9. HS-200 Histo-Clear HS-202 Histo-Clear II 無毒 + Biodegradable 使用 Histo-Clear 的客戶較多 成份略不同 Histo-Clear II 較無侵蝕性 , 但用以替換 xylene 可能需 較長的 incubation time Histo-Clear II 需與特定的 mounting medium 搭配使用 , ex. Omnimount(HS-106) HS-100 Histosol Flash point 較 xylene 高
  • 10. 浸潤 Infiltration 包埋 Embedding 包埋劑呈液態時可經由置換作用 而進入組織間隙 , 爾後凝固以便 切片 常用之包埋劑 : 石蠟 (Paraffin) 賽勞定 (Celloidin)
  • 11.
  • 12.
  • 13. 切片 Sectioning 石 臘 旋 轉 15-25μm 式 ↓ 切 3-5μm 片 機
  • 14. 染色 Staining Antigen Retrieval Protocol 裡的 xylene 亦可用 Histo-Clear 取代 !
  • 15. 染劑 Stains HS-504 Alcian Blue acid mucopolysaccharides 酸性粘多醣體 ; mast cell differentiation HS-518 Basic Fuchsin 鹼性品紅 / 鹼性復紅 / 鹼性洋紅 Main ingredient of Schiff's reagent, a pH indicator, and stain for glycoproteins and mucopolysaccharide proteins. HS-506 Biebrich Scarlet 猩紅質 Plasma stain substituted for Acid Fuchsin in Masson's trichrome. HS-516 Fast Green FCF Fibre stain substituted for Light Green SF Yellowish in Masson's trichrome.
  • 16. 染劑 Stains HS-603 Bromophenol Blue Commonly used tracking dye in electrophoresis. HS-601 Amido Black 法醫學染色:強化微弱血跡 HS-606 Methyl Green 甲 綠 基 In conjunction with Pyronin Y will differentiate RNA and DNA. HS-607 Pyronin Y #61 In conjunction with Methyl Green will differentiate RNA and DNA. HS-525 Methylene Blue 亞 藍 甲 A sensitive stain specific for DNA and RNA.
  • 18. 封片 Mounting 封片膠 / 封蓋劑 ex. 阿拉伯膠、加拿大膠 ... Mounting Media ─ aqueous ─ non-aqueous/organic 先將滴一滴封片膠在組織中央,已 45 度角緩緩將蓋 玻片蓋上,再以鑷子輕壓將氣泡趕出
  • 19. HS-103 Histomount Classic choice in synthetic mounting media HS-106 Hydromount Nonfluorescing aqueous mounting medium Effective for frozen sections, amyloid, and immunofluorescent staining procedures May be removed by soaking the slides in warm saline HS-110 Omnimount Universally compatible synthetic mounting medium. Ideally suited for use with HistoClear II. Higher flash point and Lower toxicity than xylene based mountants Provides both a safer work environment and reduces shipping costs
  • 20. 順便一併提一下 ... Anti-Fade * 不是所有 Mounting Solution 都含有 Anti-Fade!* ReadiUse™ Microscope Mounting Solution ( 編號 20009) FluoroQuest™ Anti-fading Kit I *for Slide* ( 編號 20001) FluoroQuest™ Anti-fading Kit II *for Plate* ( 編號 20003) AAT : 20009 不含 Anti-Fade 成份 Fluoromount( 編號 NB900-66726)( 含 Anti-Fade) We do not add anti-fading compounds to our media in order to allow the maximum flexibility in their use, but they are compatible with all the common anti-fade additives- your customer can add, for example, DABCO to Hydromount, and achieve excellent result. i-BRITE Plus( 編號 SF40000-10)( 含 Anti-Fade) Mounting Medium, Aqueous, BioAssay™ ( 編號 M4679-01) Mounting Medium, Organic, BioAssay™ ( 編號 M4679) USB : There is no antifade added to either M4679 or M4679-01.
  • 21. 參考連結 Tissue Preparation for Light Microscopy http://www.youtube.com/watch?v=TLm37BbR1mo Protocols Online | Archive | Histology http://protocolsonline.com/category/histology/ 獸醫科技資訊網:參考手冊 動物傳染病實驗室檢驗方法 總論 第 二   十 章 病 學 查   http://vettech.nvri.gov.tw/Articles/handbook/916.html 理 檢 法 Rotary Microtome Section http://www.youtube.com/watch?v=KnMdSgd5mts Immunohistochemistry http://www.youtube.com/watch?v=mpK5ojrXQs0
  • 22. 液相閃爍計數 LSC Liquid Scintillation Counting 閃爍計數器為利用某些物質所產生的閃爍光來偵 測游離輻射 其檢體測試形式可分為液體或固體 液體閃爍計數器是利用閃爍材料如螢光劑將放射 性同位素之輻射能轉變為光能再由光電管將光 能轉為電脈衝進而測定輻射線的數量及能量 成大 - 藥理學研究所 - 公用儀器 -B- 計數器說明 http://www.ncku.edu.tw/~pharma/v2011/8-2.php
  • 23. 液相閃爍計數 LSC Liquid Scintillation Counting Cocktails ─ 1.Solvent 溶解度 collector & conductor of energy must not quench Scintillator 2.Scintillator - primary 能被 excited solvent molecules 激發 - secondary wavelength shifters( 早期 ) improve efficiency( 現在 )
  • 25. 液相閃爍計數 LSC Liquid Scintillation Counting - Metabolic studies - Protein and DNA analysis studies 細胞增生實驗 (3H-Thymidine Incorporation/Cell Proliferation Assay) 利用活細胞合成 DNA 的過程,使其攝入放射性 3H-Thymidine , 依其放射強度代表細胞數目多寡 一排一排地將細胞 收集 (Cell Harvester) 至濾紙 ( 玻璃纖維 ) 上, 然後將含有 DNA 的濾紙剪下,置入計測瓶內,每個計測瓶加 1.5 - 2 ml 閃爍計數液,置入 Beta counter 內計測
  • 26. 液相閃爍計數 LSC Liquid Scintillation Counting 細胞收集器 (Cell Harvester)
  • 27. 液相閃爍計數 LSC Liquid Scintillation Counting 液態閃爍計數器 (Liquid Scintillation Analyzer, β-Counter) 國立陽明大學 高雄醫學大學 臺灣大學 國衛院育成中心 中興大學 東海大學 中山醫學大學 中臺科技大學 屏東科技大學 ...
  • 28. 液相閃爍計數 LSC Liquid Scintillation Counting Scintillation Vials Precision Molded Highest Quality HDPE Leak-Tight Seal Economical SVC-20 20 ml SVC-08 8 ml SVC-06 6 ml
  • 29. 閃爍計數液 LSC Cocktails The Waste Disposal Issues       in Scintillation Counting The components of the LSC cocktail may represent a hazard or a disposal problem in addition to the radioactivity. For many experiments, only a small percentage of the samples counted will have significant radioactivity, so disposal of the LSC is the predominating issue. Fortunately, biodegradable LSC cocktails have been developed, such as N.D.s' Ecoscint fluids and Uniscint BD, which substantially reduce the difficulty of disposing of LSC waste.
  • 30. 閃爍計數液 LSC Cocktails LS-273 Ecoscint A The Ideal Universal Scintillation Cocktail All Around Performance High Efficiency with High Sample Holding Biodegradable, Reduced Toxicity Solvent Biodegradable High Flash Point (>150°C) Exce ent for: ll Agar e gels, aqueous sa os mples, biologica samples, environmental samples, glass l fibe or paper filters plasma, polyacry mide gels, salt solutions, sucrose, r , la solu ized tissues. bl Useful for : Acidic sam es, alkaline sam es, catecholamine assays, high pl pl salt solut ns, tissue homog ates. io en
  • 31. Fin.