Wako 031-17601 胶原酶 I型 Collagenase Type I

Wako 031-17601 胶原酶 I型 Collagenase Type I

【产品名称】Collagenase Type I

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药

【制造商】Wako Pure Chemical Industries, Ltd.

【销售商代码】031-17601

【规格】100mg

【CAS.NO】9001-12-1

【保存条件】Keep at RT.

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

【外观】Brown, crystalline powder – powder

【注意事项】This product is for research use only. Do not administer it to human.

Source: Clostridium histolyticum

Solubility: The 1 mg is dissolved in 1 mL of water.

This is an enzyme that specifically hydrolyzes collagen as a substrate

and the activities of collagenase, caseinase, clostripain and trypsin are contained in good balance. It is mainly used for isolation of cells from epithelial tissue, lung and adipose tissue.

ES·iPS Catalog 2013

产品描述

胶原酶(Collagenase)是一种蛋白酶,一种肽链内切酶,能够特异性的识别Pro-X-Gly-Pro序列(该序列高频率出现在胶原中,很少发现于其他蛋白中)并切割该序列中性氨基酸(X)和甘氨酸(Gly)之间的肽键。许多蛋白酶都能水解单链且变性的胶原多肽,但胶原酶是唯一一种可以降解具有三股超螺旋结构的天然胶原纤维的蛋白酶,这种胶原纤维广泛存在结缔组织内。

本品是一种酶粗提物,不仅含有胶原酶(更准确的称法为梭菌蛋白酶A(clostridiopeptidase A)),能够降解天然胶原和网状纤维。还含有其他的一些蛋白酶、多糖酶、脂酶等,分别能够有效水解结缔组织和上皮组织细胞外基质内的其他蛋白,多糖和脂质,使得本品非常适用于组织消化。

目前商业化提供的细菌胶原酶主要根据胶原酶活性的差异,分为四种类型:胶原酶I型,II型,III型和IV型,在应用上有所偏向:1)胶原酶I(Type I Collagenase):含有比较均匀的各种酶活力(包括胶原酶、酪蛋白酶、梭菌蛋白酶、胰蛋白酶活性)。通常用作上皮细胞、肝、肺、脂肪和肾上腺组织细胞的制备;2)胶原酶II(Type II Collagenase):含有更高的梭菌蛋白酶活性,通常用于心脏、骨、肌肉、胸腺和软骨等组织来源细胞的制备;3)胶原酶III(Type III Collagenase):含有较低的蛋白酶活性,常用于乳腺细胞的制备;4)胶原酶IV(Type IV Collagenase):含有低胰酶活性,通常用于胰岛细胞的制备,或者需要维持受体完整性的细胞制备实验。

本品为I型胶原酶,可用于上皮、肝、肺、脂肪和肾上腺等组织和细胞的解离。

酶活力单位定义

在37℃,pH7.5 的条件下,5h内水解胶原产生相当于1µM L-亮氨酸的酶量定义为1个酶活力单位。

运输和保存方法

室温运输。4℃避光保存,2年有效。储存液-20℃避光冻存。

注意事项

为了您的安全和健康,请穿实验服并戴一次性手套操作。

使用方法

  1. 胶原酶储存液的配制

向每管100mg的胶原酶中加入100µL的含Ca2+、Mg2+的HBSS(Hank’s平衡盐溶液,含Ca2+、Mg2+),轻轻旋涡震荡使其充分溶解,制备成1g/ml(即1000×)的储存液。然后用低蛋白结合性的0.22μm的滤膜过滤除菌,分装成小份量,然后于-20℃避光冻存。

使用前于冰上解冻,避免反复冻融。其用于组织和细胞分散的常用浓度为:0.5-2.5mg/ml,用于软骨消化的常用浓度为1-2mg/ml,需要根据特定的实验条件或者参考相应的文献资料确定所需的最佳工作浓度。

2.组织的分离

1)使用无菌手术刀或剪刀将组织切成3-4mm大小的组织块;

2)利用含Ca2+、Mg2+的HBSS洗涤组织块数次;

3)加入足量的含Ca2+、Mg2+的HBSS,使其浸没组织块,并加入胶原酶至需要工作浓度;

4)于37℃孵育4-18h。消化时使用水平摇床以及用3mM的CaCl2补充消化可以提高消化效率。

5) 已分散开的细胞可使用不锈钢或尼龙网筛筛得,收集备用。未完全解离的组织另外添加适量的新鲜胶原酶工作液于37℃继续孵育;

6)利用不含胶原酶的HBSS洗涤收集的细胞数次;

7)细胞培养液重悬上述细胞,利用自动细胞计数器或其他方法计算活细胞密度。

8)于细胞培养皿上利用合适细胞培养基接种细胞。

3. 器官灌注

1)向37℃预热的含Ca2+、Mg2+的HBSS中加入胶原酶,另添加3mM的CaCl2有助于提高分离效率;

2)按照已优化的速率对相应的器官灌注胶原酶工作液;

3)将上述过程中回收的灌注液流经不锈钢或尼龙网筛,从而将已解离的细胞或小片段组织块与较大团块分离开来,未充分解离的组织需利用新鲜胶原酶工作液于37℃进一步孵育;

4)利用不含胶原酶的HBSS洗涤收集的细胞数次;

5)细胞培养液重悬上述细胞,利用自动细胞计数器或其他方法计算活细胞密度。

6)于细胞培养皿上利用合适细胞培养基接种细胞。

 

 

货号   品名     规格  Cas

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

012-01965   Alminium Oxide   500g     –

012-03305   Ammonium Dihydrogenphosphate  500g     7722-76-1

012-08643   Amino Acids Mixture Standard Solution, Type B 1mL×5A –

013-08391   Amino Acids Mixture Standard Solution, Type H 5mL      –

015-14461   Amino Acids Mixture Standard Solution, Type AN-2  5mL      –

015-25191   Anti Phosphorylated α-Synuclein, Monoclonal Antibody (pSyn#64)      50ul      –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

011-14463   Amino Acids Mixture Standard Solution, Type AN-2  1mL×5A –

011-19365   2,2′-Azobis[2-(2-imidazolin-2-yl)propane]

Dihydrochloride   500g     27776-21-2

016-08641   Amino Acids Mixture Standard Solution, Type B 5mL      –

016-20001   Anti Iba1, Rabbit (for Western Blotting)     50μg     –

016-23281   Anti Rat P2X4, Monoclonal Antibody     50ug     –

017-19362   2,2′-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride    25g 27776-21-2

018-08721   Agar, Powder     100g     9002-18-0

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

018-22021   Anti Mouse Ago2, Monoclonal Antibody 100μL    –

019-03435   Ammonium sulfate    500g     7783-20-2

019-19741   Anti Iba1, Rabbit (for Immunocytochemistry)    50μg     –

019-22291   Anti Olfactory Marker Protein, Goat      100μL    –

021-02195   Boric acid     500g     10043-35-3

021-16201   BES-H2O2(Cell-impermeant)   1mg      –

031-17601   Collagenase Type I    100mg   9001-12-1

032-08385   Citric Acid Monohydrate   500g     5949-29-1

032-15372   Casein Phosphopeptide    25g –

034-06363   N-Caprylic Acid   25mL    124-07-2

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

034-16111   Cefotaxime Sodium Salt   1g   64485-93-4

035-04595   Creatinine    500g     60-27-5

035-17604   Collagenase Type I    1g   9001-12-1

038-23221   α-Casein, from Bovine Milk, Dephosphorylated  1mg      –

039-01335   Carboxymethyl Cellulose Sodium Salt   500g     9004-32-4

021-17801   BES-So-AM 1mg      936356-51-3

022-07985   γ-Butyrolactone  500mL   96-48-0

025-03195   Buffer Solution Standard (Phosphate pH Standard Equimolal Solution)

pH6.86 (25 degrees C)    500mL   –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

047-18863   Dexamethasone 100mg   50-02-2

049-18801   4′,6-Diamidino-2-phenylindole Dihydrochloride n-Hydrate      10mg      28718-90-3

058-07681   EasySeparator    1set      –

025-03195   Buffer Solution Standard (Phosphate pH Standard Equimolal Solution)

pH6.86 (25 degrees C)    500mL   –

019-08393   Amino Acids Mixture Standard Solution, Type H 5Ax1mL –

019-11941   Active Carbon-impregnated Silica Gel   10g –

019-19741   Anti Iba1, Rabbit (for Immunocytochemistry)    50μg     –

025-15481   BES-Thio     1mg      –

026-16371   BF-170  1mg      –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

027-01276   Benzyl Alcohol    500mL   100-51-6

028-03185   Buffer Solution Standard (Phthalate pH Standard Solution)

pH4.01 (25 degrees C)    500mL   –

028-03185   Buffer Solution Standard (Phthalate pH Standard Solution)

pH4.01 (25 degrees C)    500mL   –

028-03205   Buffer Solution Standard (Tetraborate pH Standard Solution)

pH9.18 (25 degrees C)    500mL   –

077-03155   Gelatin   500g     9000-70-8

081-00136   Heparin Sodium  10mu    9041-08-1

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

088-00705   L-Histidine Hydrochloride Monohydrate      500g     5934-29-2

093-06471   Insulin, Human, recombinant   50mg    –

097-06474   Insulin, Human, recombinant   1g   11061-68-0

120-04891   L-012    100mg   –

125-05061   Lysyl Endopeptidase, Mass Spectrometry Grade      5×20μg  –

125-05061   Lysyl Endopeptidase, Mass Spectrometry Grade      5×20μg  –

125-05061   Lysyl Endopeptidase, Mass Spectrometry Grade      5×20μg  –

129-02541   Lysyl Endopeptidase( R)   10AU     –

129-02541   Lysyl Endopeptidase( R)   10AU     –

129-02541   Lysyl Endopeptidase( R)   10AU     –

129-04861   LY 294002   5mg      154447-36-6

130-07871   Melibiose     5g   585-99-9

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

028-16211   BES-So (Cell-impermeant)      1mg      –

028-17811   BES-H2O2-Ac     1mg      –

029-16361   BF-168  1mg      –

039-23511   CultureSure? Freezing Medium    100ml    –

041-18861   Dexamethasine   1g   50-02-2

042-29445   Dibasic Sodium Phosphate Hydrate 500g     10039-32-4

043-22611   Dextran 200,000 100g     9004-54-0

064-05381   Fibroblast Growth Factor (basic)(FGFb / bFGF / FGF2),

Human, recombinant, Animal-derived-free  50ug     106096-93-9

071-02293   gamma-Globulin, from Human Serum   5g   9007-83-4

075-03075   Gellan Gum  500g     71010-52-1

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

131-00405   Magnesium Sulfate    500g     10034-, 99-8

136-03032   Methyl Red   25g 493-52-7

137-06085   Molecular Sieves 4A1/16  500g     70955-01-0

138-07872   Melibiose     25g 585-99-9

145-08221   Nuclease P1 500u     54576-84-0

145-08221   Nuclease P1 500u     54576-84-0

162-09115   Polyethylene Glycol 4,000 500g     25322-68-3

163-03545   Potassium Chloride    500g     7447-40-7

164-21655   N/400 Potassium Polyvinyl Sulfate Solu 500mL   26837-42-3

191-01785   Trisodium Citrate Dihydrate    500g     6132-04-3

193-02041   Glycerol 2-Phosphate Disodium Salt n-Hydrate   100g     819-83-0

195-12854   Streptavidin, from Streptomyces avidinii     5mg      9013-20-1

195-16031   StemSure (R) Freezing Medium     100ml    –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

196-00015   Sucrose 500g     57-50-1

196-08675   Sodium Dodecyl Sulfate    500g     151-21-3

197-07485   Sodium Sulfate   500g     7757-82-6

199-02825   Sodium Dihydrogenphosphate Dihydrate    500g     13472-35-0

203-17561   Trichostatin A     1mg      58880-19-6

207-00055   L(+)-Tartaric Acid     500g     87-69-4

208-08385   1mol/l Triethylammonium Hydrogencarbonate Solution   500mL   15715-58-9

223-02112   VA-044  25g 27776-21-2

225-02111   VA-044  100g     27776-21-2

165-09105   Polyethylene Glycol 2,000 500g     25322-68-3

165-17035   Polyvinylpyrrolidone K 30 500g     9003-39-8

167-09045   Polyethylene Glycol 200   500g     25322-68-3

169-04245   Potassium Dihydrogen Phosphate  500g     7778-77-0

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

169-09125   Polyethylene Glycol 6,000 500g     25322-68-3

185-01003   Rifampicin    5g   13292-46-1

186-01114   all-trans-Retinoic Acid 50mg    302-79-4

189-01001   Rifampicin    1g   13292-46-1

190-05535   Trisodium Citrate Dihydrate    500g     6132-04-3

191-01665   Sodium Chloride  500g     7647-14-5

227-01071   Valproic Acid 5g   99-66-1

231-02011   Wetting Tension Test Mixture No.52.0  50mL    –

232-01941   Wetting Tension Test Mixture No.42.0  50mL    –

233-01971   Wetting Tension Test Mixture No.45.0  50mL    –

234-02001   Wetting Tension Test Mixture No.50.0  50mL    –

235-01931   Wetting Tension Test Mixture No.41.0  50mL    –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

235-02031   Wetting Tension Test Mixture No.56.0  50mL    –

236-01961   Wetting Tension Test Mixture No.44.0  50mL    –

238-01921   Wetting Tension Test Mixture No.40.0  50mL    –

238-02021   Wetting Tension Test Mixture No.54.0  50mL    –

238-02141   Wetting Tension Test Mixture No.73.0  50mL    7732-18-5

253-00513   Y-27632 5mg      331752-47-7

257-00511   Y-27632 1mg      331752-47-7

290-34251   Presep-C DNPH   20EA     –

296-63801   LabAssay? Phospholipid   1300tests     –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

298-64101   Fructooligosaccharides standard set     set  –

302-93561   Phos-tag (TM) Agarose    0.5mL    –

304-93521   Phos-tag (TM) Acrylamide AAL-107      10mg    –

304-93521   Phos-tag (TM) Acrylamide AAL-107      10mg    –

290-34251   Presep-C DNPH   20EA     –

290-63701   LabAssay? Triglyceride    1000tests     -2016-2-29

290-65901   LabAssay? Creatinine      500tests –

291-58601   Lab Assay ALP    900tests –

293-51601   Endotoxin Extracting Solution for LAL Test  10mL x 4     –

294-62001   DsDD cDNA Subtraction kit Wako   5tests    –

294-63601   LabAssay? NEFA 750tests –

294-65801   LabAssay? Cholesterol    1000tests     –

295-50201   DNA Extractor? Kit   50tests  –

295-51301   Limulus ES-II Single Test Wako     25 tests –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

295-51301   Limulus ES-II Single Test Wako     25 tests –

295-73401   Fructooligosaccharides Set      Set  –

304-93526   Phos-tag (TM) Acrylamide AAL-107 5mM Aqueous Solution   0.3ml    –

305-93551   Phos-tag? Mass Analytical Kit set  –

308-93541   Phos-tag (TM) Biotin BTL-105 10mg    –

308-93563   Phos-tag (TM) Agarose    3mL      –

308-97201   Phos-tag Biotin BTL-1111 1mM Aqueous Solution    0.1000mL    –

325-88521   Lipase PS Amano SD  10g –

298-65701   LabAssay? Glucose   1000tests     –

300-93523   Phos-tag (TM) Acrylamide AAL-107      2mg      –

301-93531   Phos-tag (TM) Biotin BTL-104 10mg    –

301-99413   Phos-tagTM Biotin BTL-104 1mM Aqueous Solution[for Campaign]     0.1mL      753451-66-0

302-93522   Phos-tagTM Acrylamide AAL-107 5mM Aqueous Solution[for Campaign]      0.3mL    871839-54-2

631-00651   Cellmatrix Type I -A (Collagen, Type I, 3 mg/mL, pH 3.0)     20mL    –

Wako  031-17601  Collagenase Type I 9001-12-1   和光纯药 现货

637-00653   Cellmatrix Type I -A (Collagen, Type I, 3 mg/mL, pH 3.0)     100mL   –

637-00653   Cellmatrix Type I -A (Collagen, Type I, 3 mg/mL, pH 3.0)     100mL   –

638-01021   Endotoxin Standad /JPSE  10000un      –

986-10001   Anti asialo GM1 (Rabbit), (014-09801)  1mL      617-45-8

Research Diets官网代理商 动物饲料代理商

特色

Research Diets官网代理商 动物饲料代理商

美国research diets公司是一家专业生产实验室动物饲料的大型公司,包括脂肪热能的颗粒饲料及各种维生素缺乏饲料的专业供应商,Research Diets是生产动物实验用高脂肪饲料的先驱,可配制高达 60 kcal% 的高脂肪饲料,成份內含各式脂肪及碳水化合物,Research Diets长期陪伴实验室科研人员,以配制研究需要的饲料配方,可用于诱发肥胖病及糖尿病的特殊饲料如D12450B、D12451及 D12492, Research Diets拥有配方饲料种类超过15000种,常规高端饲料10多种如AIN-76A Western饲料等
食源性肥胖症饲料    

Research Diets动物造模饲料Research diets 食源性肥胖症饲料

肥胖是一种世界范围内的慢性疾病,是导致高血压、糖尿病、冠心病、心肌梗死等多种慢性病发生的主要危险因。

 

遗传和环境因素对肥胖症的发生起重要作用,膳食则是影响肥胖症的主要的环境因素之一。膳食诱导的肥胖动物模型普遍被用于肥胖发生机理和治疗方法的研究。

 

我们与世界顶级动物饲料供应商美国RDI合作,为客户提供配方可报告、可复制、可修改的纯化饲料,我们的饲料具有造模时间短、造模效果好、有效易复制等特点。

 

重点推荐饲料:D12492和D12451。

 推荐产品  货号  规格  生产厂商
60%Kcal High-Fat(DIO) Diets D12492 12.5kg 美国RDI
45%Kcal High-Fat(DIO) Diets D12451 12.5kg 美国RDI
10%Kcal High-Fat(DIO) 7% Sucrose Control Diets match D12492 D12450J 12.5kg 美国RDI
10%Kcal High-Fat(DIO) 17% Sucrose Control Diets match D12451 D12450H 12.5kg 美国RDI
10%Kcal High-Fat(DIO) 35% Sucrose Control Diets D12450B 12.5kg 美国RDI
10%Kcal High-Fat(DIO) No Sucrose Control Diets D12450K 12.5kg 美国RDI

Wako SeeDB实现生物体样本深层成像


产品编号 产品名称 产品规格 产品等级 产品价格
291-79601 SeeDB实验试剂盒
 SeeDB Trial Kit
1kit 组织透明化
193-18391 SeeDB:20w/v% 果糖溶液
 SeeDB:20w/v% Fructose Solution
250mL 组织透明化
196-18401 SeeDB:40w/v% 果糖溶液
 SeeDB:40w/v% Fructose Solution
250mL 组织透明化
193-18411 SeeDB:60w/v% 果糖溶液
 SeeDB:60w/v% Fructose Solution
250mL 组织透明化
190-18421 SeeDB:80w/v% 果糖溶液
 SeeDB:80w/v% Fructose Solution
250mL 组织透明化
197-18431 SeeDB:100w/v% 果糖溶液
 SeeDB:100w/v% Fructose Solution
250mL 组织透明化
194-18441 深层透明化试剂
 SeeDB
250mL 组织透明化

Wako SeeDB实现生物体样本深层成像SeeDB

实现生物体样本深层成像

  今井猛博士等人开发了一种对于水溶性生物体组织的形态和组成在不破坏荧光蛋白以及荧光神经同位素的条件下,可快捷简便地使大脑等生物体组织透明化的方法——SeeDB(See Deep Brain)。SeeDB由水、果糖、还原剂构成。

  SeeDB法是在使用共聚焦显微镜和双光子激发显微镜下可深层成像的方法。可利用荧光蛋白和神经示踪剂,实现对荧光神经回路的全貌阐明和定量分析等各种各样的应用。


详细内容,请点击SeeDB技术开发者今井猛博士的网站:SeeDB Resources(本部分内容亦可参见相关资料

◆SeeDB protocol案例


  SeeDB法,可用于脊椎动物的各种组织,下面以小鼠大脑为例进行介绍。


1.固定

 将小鼠大脑在4%多聚甲醛/PBS,4℃下固定过夜。

 样本用PBS清洗三次(每次清洗10分钟)


2.透明化处理

③ 将样品加入放有20 mL的SeeDB:20 w/v%果糖溶液的50 mL锥形瓶中。

  在室温下在旋转器中旋转4-8小时(约4 rpm)

  在脆弱的样本和薄片情况也可以用压板式摇床(约17 rpm)

④ 将样本加入放有SeeDB:40 w/v%果糖溶液的50mL锥形管中,室温下旋转4-8小时。

⑤ 将样本加入放有SeeDB:60 w/v%果糖溶液的50mL锥形管中,室温下旋转4-8小时。

⑥ 将样本加入放有SeeDB:80 w/v%果糖溶液的50mL锥形管中,室温下旋转12小时。

⑦ 将样本加入放有SeeDB:100 w/v%果糖溶液的50mL锥形管中,室温下旋转12小时。

⑧ 将样本加入放有SeeDB的50 mL锥形管中,室温下旋转24小时。

    最长时间可延长至48小时。在透明化成功时,透光可观察到组织透明化。


3.观察

⑨ 将SeeDB处理过的大脑样本置于共聚焦显微镜或双光子激发显微镜下观察。


(注意点)

  样本用SeeDB液固定。用SeeDB透明化处理后的样本折射率可达到1.49。因此,物镜中应该有甘油浸没透镜、油镜、透明化用镜片,即使浸水镜头到达2 mm深度也是没有问题的。浸没式镜片请使用浸没液,SeeDB不能用作浸没液。在使用干式镜头(折射率1.0)和浸水镜头(折射率1.33)获取画像情况下,需要补正深度值(补正值请参考相关文献)。

 


SeeDB 处理案例

Wako SeeDB实现生物体样本深层成像

SeeDB生物体样本透明化

左起依次为经SeeDB液透明化处理的小鼠幼胎(幼胎12天)

新生鼠(生后3天)的全脑、成鼠大脑(8周龄,厚度2 mm)


SeeDB 观察案例

Wako SeeDB实现生物体样本深层成像

Wako SeeDB实现生物体样本深层成像

双光子激发显微镜下的Thy1-YFP-H小鼠

(10周龄)大脑荧光成像

使用Olympus公司产品 

多光子专用物镜:XLPLN10XSVMP观察案例

共聚焦显微镜下的Thy1-YFP-H小鼠

(10周龄)大脑荧光成像

使用Olympus公司产品

有机硅浸没式物镜:UPLSAPO 10X2观察案例

Wako SeeDB实现生物体样本深层成像

双光子激发显微镜下的Thy1-YFP-H小鼠

(10周龄)大脑荧光成像

使用Olimpus公司产品

多光子专用物镜:XLSLPLN25XGMP观察案例

比例尺:100 μm

Wako SeeDB实现生物体样本深层成像

小鼠大脑固定后用Dil标记,SeeDB透明化处理案例   

左:嗅球僧帽细胞树突(横向观察)

右:嗅球僧帽细胞树突(纵向观察)

使用Olympus公司产品

有机硅浸没型物镜:UPLSAPO 20X观察案例。比例尺:100 μm。


组织透明化配套耗材:成像用透明室

Wako SeeDB实现生物体样本深层成像

透明化试剂方法选择流程.pdf

组织透明化试剂Q&A ver1.0

Tissue clearing reagent Q&A ver1.0

Q组织透明化的原则

A:组织透明化通常是按下面的步骤进行的:

A:(a)固定 (b)透化 (c)脱色 (d)折射率匹配

 

Q:组织透明化的优势

A:目前大多数可用的透明化技术,都是通过编码荧光报告蛋白或者用荧光标记抗体进行后标记,从而可视化

A:组织中特定的蛋白。组织透明化成为了具有单细胞分辨率的组织3D成像中重要的一环。

 

Q:透明化技术的比较

A:下面是水性的透明化技术。

Wako SeeDB实现生物体样本深层成像


Wako SeeDB实现生物体样本深层成像

A:SeeDB(基于果糖的方法)在一些应用中有一定优势。因为SeeDB不含去垢剂,所以对于透明化DiI(细胞膜

A:红色荧光探针,一种神经示踪剂)标记的样品和使用光电关联显微镜(CLEM)的情况下,SeeDB有着一定优

A:势。另外SeeDB会产生自体荧光,有时候会对辨认未标记的神经元结构(如神经毡)起着一定帮助。

A:SeeDB2是为使用高NA物镜的高分辨率成像而优化的技术;因此,当处理较大的组织时,CLARITY或者CUBIC

A:会是更好的选择。

 

Q:切片厚度的最大值和最小值?

A:您应该根据您的物镜的WD(工作距离)来决定切片的厚度。我们建议切片的厚度不要超过3mm。对于十分薄

A:且脆弱的样品,我们建议使用琼脂糖包埋。

A:使用CUBIC,可以让全器官、全身透明化和使用LSFM的快速3D成像具有可重复性。

 

Q:试剂盒成分

A:具体如下:

A:1. SCALEVIEW-S试剂盒(299-79001)适用于20份1-2mm的切片样品

A:A:1) SCALEVIEW-S0——100mL

A:A:2) SCALEVIEW-S1——100mL

A:A:3) SCALEVIEW-S2——100mL

A:A:4) SCALEVIEW-S3——100mL

A:A:5) SCALEVIEW-S4——100mL

A:A:6) SCALEVIEW-SMT——100mL

 

A:2. SCALEVIEW-S试剂盒(290-80801)适用于20-30份1-2mm的切片样品。

A:A:1) SCaleCUBIC-1 Solution——250mL

A:A:2) SCaleCUBIC-2 Solution——250mL

A:A:3) Mounting Solution 1——40mL

A:A:4) Mounting Solution 2——40mL

 

A:3. SeeDB试剂盒(299-79901)适用于20-30份1-2mm的切片样品。

A:A:1) SeeDB:20w/v% Fructose Solution——50mL

A:A:2) SeeDB:40w/v% Fructose Solution——50mL

A:A:3) SeeDB:60w/v% Fructose Solution——50mL

A:A:4) SeeDB:80w/v% Fructose Solution——50mL

A:A:5) SeeDB:100w/v% Fructose Solution——50mL

A:A:6) SeeDB——50mL

 

A:4. SeeDB2试剂盒(294-80701)适用于20-30份1-2mm的切片样品。

A:A:1) Saponin(皂角苷)——5g

A:A:2) SeeDB2G solution——50mL

A:A:3) SeeDB2S solution——50mL

A:A:4) PBS——50mL

 

Q:样品的保存

A:SCALEVIEW-S处理的样品应在室温下保存2至3周。您可以在2至3周内观察SCALEVIEW-S透明化的样品中的

A:荧光信号。

A:CUBIC处理的样品应在室温下保存1周。您可以在1周内观察CUBIC透明化的样品中的荧光信号。

A:SeeDB2G处理的样品应在冰箱中保存1年,而SeeDB2S处理的样品则可在室温下保存1年。您可以在1年内观

A:察SeeDB透明化的样品中的荧光信号。

 

Q:抗体的荧光标记

A:应在进行透明化前就进行抗体的荧光标记。

 

Q:适用于SCALEVIEW-S的显微镜

A:SD-OSR(Olympus)

A:FV-OSR(Olympus)

 

Q:适用于CUBIC的显微镜

A:SD-OSR(Olympus)

A:FV-OSR(Olympus)

A:Zeiss Lightsheet Z.1(Carl Zeiss)

 

Q:适用于SeeDB2的显微镜

A:Confocal(最好使用高NA的油浸镜头)

A:STED(Leica Microsystems)

A:SR-SIM(Carl Zeiss)

A:SD-OSR(Olympus)

A:FV-OSR(Olympus)

A:Airyscan(Carl Zeiss)

A:HyVolution(Leica Microsystems)

A:Two-photon(最好使用复合浸没镜头)

A:Confocal(最好使用高NA的甘油浸镜头)

A:Light-sheet DLS(Leica,最好使用定制镜面的设备)

 

Q:重构

A:对于定量用途,我们建议使用Neurolucida系统(MBF)。我们同样测试了VAST Lite用于成像用途时的表

A:现。重构时也可以使用开源软件。例如,Vaa3d可以处理大量的拼接图像的数据。

 

Q:物镜推荐

Wako SeeDB实现生物体样本深层成像

 

Q:其他物种?其他器官?

A:对于昆虫样品和鱼类样品,建议选择CUBIC。

A:昆虫相关文献:Ohuma.et al.: Fish Pathology, 52(2), 96(2017).

A:鱼类相关文献:Konno and S.Okazaki .: Zoological Letters, 4:13(2018).

   

  对于多细胞球状体,选择SCALEVIEW-S会更好。

  https://www.nature.com/articles/s41598-018-29169-0

 

参考文献:

[1] Ke, M. T., Fujimoto, S. and Imai, T. : Nat Neurosci ,6 (8),1154 (2013).

[2] Ke, M. T., Fujimoto, S. and Imai, T.: Bio-protocol ,4(3), e1042 (2014).

[3] Ke, M. T., and Imai, T. : Curr Protoc Neurosci ,66, 2.22.1-2.22.19 (2014).

甘油检测试剂盒 Glycerol Assay Kit 货号:K-GCROL Megazyme中文站

甘油检测试剂盒

英文名:Glycerol Assay Kit

货号:K-GCROL

规格:70 assays (manual) / 700 assays (microplate)

分析物意义:常见食品组分,或作为甜味剂,或用于改善口感

Megazyme检测试剂盒优点:新型的药片模式,性质更稳定,反应快

The Glycerol test kit is a simple, reliable, rapid and accurate method for the measurement and analysis of Glycerol in beverages, foodstuffs and other materials.
Suitable for manual and microplate formats.

UV-method for the determination of Glycerol in foodstuffs,
beverages and other materials

Principle:
(glycerokinase)
(1) Glycerol + ATP → L-glycerol-3-phosphate + ADP

(pyruvate kinase)
(2) ADP + PEP → ATP + pyruvate

(L-lactate dehydrogenase)
(3) Pyruvate + NADH + H+ → L-lactic acid + NAD+

Kit size: 70 assays (manual) / 700 (microplate)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 5 min
Detection limit: 0.34 mg/L
Application examples:
Wine (and grape juice), beer, spirits, vinegar, marzipan, fruit juices,
soft drinks, toothpaste, honey, tobacco, paper (and cardboard),
cosmetics, pharmaceuticals, soap and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by OIV and
MEBAK

Advantages

  • Novel tablet format for increased stability
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years as supplied
  • Very rapid reaction
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual and microplate formats

 

 Q1. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q2. Is K-GCROL specific for glycerol?

K-GCROL is highly specific for glycerol.
Some compounds that are known not to react or interfere with the assay include:
Polyethylene glycol
Ethylene glycol  
Propylene glycol 

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q5. Is the Glycerol Assay Kit (K-GCROL) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed, by dilution (if required) in distilled water. 

Q6. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q7. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q8. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q9. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q10. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q11. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q12. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q13. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q14. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q15. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q16. To measure fermentation samples contain high microbial cell density, is cell disruption required?

Cell disruption is only require to measure glycerol within microbial cells.

To measure glycerol in the extracellular media only then cell disruption is not required and centrifugation of the sample may be sufficient, e.g.:

(a) Determination of glycerol in cell culture media/supernatants. In general, the concentration of glycerol in cell culture media/supernatants can be determined without any sample treatment (except clarification by centrifugation/filtering or dilution according to the dilution table, if necessary). Typically, no clarification or dilution is required, and a sample volume of 0.1 mL is satisfactory.

If interference is suspected then sample clarification/deproteinisation using carrez reagents or perchloric acid should be used (methods are provided in the kit booklet).