Vector Laboratories’ Antigen Unmasking Solutions are highly effective at revealing antigens in formalin-fixed, paraffin embedded tissue sections when used in combination with a high temperature treatment procedure. This citrate-based solution (H-3300) is pH 6.0 and is supplied as convenient 100x concentrated stock, sufficient for preparation of 25 L of working solution.
Advanced avidin/biotin technology: The Elite® ABC complex is smaller, very uniform, and highly active, allowing more accessibility for binding to a biotinylated target
HIghest sensitivity, low background: The VECTASTAIN® Elite® ABC system is the most sensitive avidin/biotin-based peroxidase system. The Elite® ABC series is approximay 5 times more sensitive than the original VECTASTAIN® ABC Kit with the same low background.
Cost effective: HIgher sensitivity leads to lower cost per slide
Available without (“Standard” kit) or with biotinylated species-specific or universal secondary antibodies
Available in ready-to-use formats: Prediluted, stabilized working solutions of Elite® ABC Kit reagents provide the same high sensitivity and low background as the traditional VECTASTAIN® Elite® ABC Kit reagents.
Vector防荧光萃灭封片剂H-1200 VECTASHIELD Mounting Medium w
产品型号： H-1200
简单描述
Vector防荧光萃灭封片剂H-1200 VECTASHIELD Mounting Medium with DAPI产品名称：VECTASHIELD含有DAPI的封片剂别名：VECTASHIELD Mounting Medium with DAPI 美国Vector labs产品货号|编号:Vector labs H-1200
详细介绍
Vector防荧光萃灭封片剂H-1200 VECTASHIELD Mounting Medium with DAPI 产品名称：VECTASHIELD含有DAPI的封片剂 别名：VECTASHIELD Mounting Medium with DAPI 美国Vector labs产品货号|编号:Vector labs H-1200
Vector防荧光萃灭封片剂H-1200 VECTASHIELD Mounting Medium with DAPI
Vector H-1500: HardSet Mounting Medium with DAPI， 400张/10ml 凝固性；防荧光萃灭；含DAPI，可衬染细胞核，用于ISH或衬染DNA的实验；适于油镜观察。 FITC,Texas Red,含DAPI Rhodamine等。
Vector H-5000: Permanent Mounting Medium ,240张/60ml 非水溶性，无毒、无味；清晰；和底物BCIP/NBT联用，可将结晶减至zui小。长期保存。 HRP和AP的底物。
Vector H-5501: AQ Aqueous Mounting Medium ,240张/60ml 水溶性，适于反应产物为酒精或其它有机溶剂溶性的底物。 大多数HRP和AP的底物如AEC，DAB, BCIP/NBT, Vector SG.
ith DAPI
详细信息：
VECTASHIELD? Mounting Media are unsurpassed in preventing photobleaching. The different formulations of VECTASHIELD? Mounting Media all offer the same outstanding anti-fade and anti-photobleaching properties. They are all compatible with fluorescein, Texas Red?, AMCA, DyLight? dyes, Alexa Fluor? dyes, fluorescent nuclear stains, fluorescent proteins, fluorescent tracers, histochemical stains, and most fluorochromes.
Features of VECTASHIELD? Mounting Media:
Inhibit photobleaching of most fluorochromes
Available in non-hardening or hardening formulations
Available with or without DAPI or propidium iodide
No warming necessary
Slides can be viewed after prolonged storage
Continues to inhibit photobleaching after prolonged storage of mounted slides
Easy to use
Optically clear
Ideal refractive index
The original VECTASHIELD? Mounting Medium is a glycerol-based, aqueous mountant that does not solidify but remains a viscous liquid on the slide. After mounting, coverslipped slides will not readily dry out and can be reviewed for weeks afterwards without sealing. For prolonged storage, coverslips can be permanently sealed around the perimeter with nail polish. Mounted slides should be stored at 4 oC.
Both the VECTASHIELD? HardSet? and the original VECTASHIELD? Mounting Media are available with or without the counterstain DAPI （4’, 6-diamidino-2-phenylindole）. DAPI produces a blue fluorescence when bound to DNA with excitation at about 360 nm and emission at 460 nm.
VECTASHIELD? Mounting Media are compatible with a wide array of fluorochromes, enzymatic substrates, and fluorescent proteins. Please consult the compatibility table （located in product information box） to determine if VECTASHIELD? will be compatible in your system.
Please note: VECTASHIELD? Mounting Medium may not be compatible with all enzymatic substrates or fluorescent proteins and test sections would be advisable when using anything other than commonly used fluorochromes.
The refractive index for VECTASHIELD? Mounting Medium is 1.44.
Other manufacturers measure the antifade properties of their mountants using labeled microspheres or arrayed spots. Vector Labs prefers to measure antifade properties of VECTASHIELD? mountants using frozen tissue sections immunohistochemically stained with fluorescently labeled secondary antibodies.
Antifade capability is measured using a 40x objective with real time imaging over 30 seconds of continuous exposure to the excitation illumination. Individual intensity measurements are recorded from 6 separate labeled regions and the average is calculated. The intensity after 30 second exposure is expressed as a percentage of the intensity at zero time. The values for PG are taken from the manufacturer’s published results.
Avidin and streptavidin reagents are powerful tools to detect or purify biotinylated proteins, nucleic acids, and other macromolecules.
Vector Laboratories’ enzyme-conjugated avidin and streptavidin are produced with the highest specific activity enzymes in optimal ratios. Specific covalent linkages are chosen to provide stable, highly active conjugates.
These enzyme conjugates are suitable for use in solid-phase assays, tissue/cell staining systems, and blotting applications.
Horseradish Peroxidase Streptavidin is produced by our own coupling procedure which preserves the high specific activity of the peroxidase. Using biotinylated primary or secondary intermediates and peroxidase-labeled streptavidin, antigens can be localized in histological sections, cytospin preparations, or smears. In addition, sensitive detection systems for a variety of solid-phase assays utilizing microtitration plates or nitrocellulose have been developed using this conjugate. Horseradish Peroxidase Streptavidin is supplied as a concentrate （1mg/ml） or as a ready-to-use （R.T.U.） stabilized solution （100 ml, 1µg/ml） in a bottle fitted with a drop dispenser top.
VECTOR SBA生物素标记的大豆凝集素SBA Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
VECTOR SBA生物素标记的大豆凝集素
isolated from Glycine max （soybean） seeds
Composed of four subunits of approximay equal size, soybean agglutinin is a family of closely related isolectins. This glycoprotein has a molecular weight of about 120,000 and an isoelectric point near pH 6.0. SBA preferentially binds to oligosaccharide structures with terminal a- or b-linked N-acetylgalactosamine, and to a lesser extent, galactose residues. Binding can be blocked by substitutions on penultimate sugars, such as fucose attached to the penultimate galactose in blood group B substance. SBA has been used in glycoprotein fractionation, histochemical applications and cell sorter analysis.
An important application for SBA is the separation of pluripotential stem cells from human bone marrow. Cells fractionated by SBA do not produce graft vs host disease and can be used in bone marrow transplantation across histocompatibility barriers （references available upon request）. It should be noted that some forms of SBA seem to be excellent in separating human cells while others are better for cells of other species.
VECTOR SBA生物素标记的大豆凝集素
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation.
Inhibiting/Eluting Sugar: 200 mM N-acetylgalactosamine
Biotinylated Pisum Sativum Agglutinin豌豆凝集素Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
Biotinylated Pisum Sativum Agglutinin豌豆凝集素
isolated from Pisum sativum （garden pea） seeds Pisum sativum agglutinin is nearly identical in structure and carbohydrate specificity to Lens culinaris agglutinin. PSA has four subunits, two of approximay 17,000 daltons and two of about 6,000 daltons. Isoelectric focusing has revealed two isolectins with isoelectric points of pH 5.9 and pH 7.0. The lectin has specificity toward a-linked mannose-containing oligosaccharides, with an N-acetylchitobiose-linked a-fucose residue included in the receptor sequence. Calcium and manganese ions are required for activity. PSA has been used to fractionate cells, to isolate glycoproteins and glycopeptides, to distinguish between normal and virally transformed cells, as a T-cell mitogen, and as an inhibitor of allograft rejection.
Biotinylated Pisum Sativum Agglutinin豌豆凝集素
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation. Inhibiting/Eluting Sugar: 200 mM a-methyl mannoside/200 mM a-methyl glucoside mixture
VECTOR生物素Jacalin Biotinylated JacalinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
VECTOR生物素Jacalin Biotinylated Jacalin
isolated from Artocarpus integrifolia （Jackfruit） seeds Jacalin is a lectin composed of four subunits, two of approximay 10,000 daltons and two of 16,000 daltons each. This 50,000 dalton glycoprotein appears to bind only O-glycosidically linked oligosaccharides, preferring the structure galactosyl （b-1,3） N-acetylgalactosamine. This structure （the so-called “T-antigen”） is the oligosaccharide to which peanut agglutinin binds. However, unlike PNA, Jacalin will bind this structure even in a mono- or disialylated form. This lectin has been used to purify human IgA, since no other human immunoglobulin class binds Jacalin （references available upon request）. The specificity of this lectin also affords the opportunity to localize or isolate glycoproteins with O-glycosidically linked oligosaccharide side chains.
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation. Inhibiting/Eluting Sugar: 800 mM galactose or 100 mM melibiose
VECTOR GNL生物素雪花莲凝集素Biotinylated Galanthus Nivalis LectinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water. If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）. 1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fix sections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present, inactivate using appropriate methods. 2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash with TPBS （PBS + 0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the sections and incubate for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase, ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red （Cat. No. SK-5100）. Rinse in tap water. 6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 ºC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）. 2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS. 3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS. 5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. For peroxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No. SK-5900）. 6. Quantify the colored reaction product by spectrophotometry. Western Blot: 1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures. 2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at room temperature. Wash with TPBS （PBS +0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate the membrane in the reagent for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™ Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB （Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity. Vector Labs offers a series of sugars that are intended for such a purpose. The lectin is diluted to a suitable working concentration in a solution containing approximay 200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
VECTOR EEL生物素大叶黄杨Europaeus凝集素Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
VECTOR EEL生物素大叶黄杨Europaeus凝集素
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water. If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）. 1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fix sections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present, inactivate using appropriate methods. 2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash with TPBS （PBS + 0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the sections and incubate for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase, ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red （Cat. No. SK-5100）. Rinse in tap water. 6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 ºC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）. 2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS. 3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS. 5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. For peroxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No. SK-5900）. 6. Quantify the colored reaction product by spectrophotometry. Western Blot: 1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures. 2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at room temperature. Wash with TPBS （PBS +0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate the membrane in the reagent for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™ Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB （Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. VECTOR EEL生物素大叶黄杨Europaeus凝集素
Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity. Vector Labs offers a series of sugars that are intended for such a purpose. The lectin is diluted to a suitable working concentration in a solution containing approximay 200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water. If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）. 1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fix sections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present, inactivate using appropriate methods. 2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash with TPBS （PBS + 0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the sections and incubate for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase, ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red （Cat. No. SK-5100）. Rinse in tap water. 6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mounting VECTOR 生物素曼陀罗凝集素Biotinylated Datura Stramonium Lectin
in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 ºC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）. 2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS. 3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS. 5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. For peroxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No. SK-5900）. 6. Quantify the colored reaction product by spectrophotometry. Western Blot: 1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures. 2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at room temperature. Wash with TPBS （PBS +0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate the membrane in the reagent for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™ Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB （Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity. Vector Labs offers a series of sugars that are intended for such a purpose. The lectin is diluted to a suitable working concentration in a solution containing approximay 200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
VECTOR BPL生物素菊紫荆凝集素Biotinylated Bauhinia Purpurea LectinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water. If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）. 1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fix sections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present, inactivate using appropriate methods. 2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash with TPBS （PBS + 0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the sections and incubate for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase, ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red （Cat. No. SK-5100）. Rinse in tap water. 6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 ºC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）. 2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS. 3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS. 5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. For peroxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No. SK-5900）. 6. Quantify the colored reaction product by spectrophotometry. Western Blot: 1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures. 2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at room temperature. Wash with TPBS （PBS +0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate the membrane in the reagent for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™ Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB （Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity. Vector Labs offers a series of sugars that are intended for such a purpose. The lectin is diluted to a suitable working concentration in a solution containing approximay 200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
VECTOR 生物素苋尾状凝集素（ACL，ACA） BIOTIN-ACLBiotinylated Amaranthus Caudatus Lectin （ACL, ACA）Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
VECTOR 生物素苋尾状凝集素（ACL，ACA） BIOTIN-ACL
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water. If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）. 1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fix sections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present, inactivate using appropriate methods. 2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash with TPBS （PBS + 0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the sections and incubate for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase, ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red （Cat. No. SK-5100）. Rinse in tap water. 6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mounting in glycerol-based mounting media. VECTOR 生物素苋尾状凝集素（ACL，ACA） BIOTIN-ACL
ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 ºC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）. 2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS. 3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to the wells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS. 5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. For peroxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No. SK-5900）. 6. Quantify the colored reaction product by spectrophotometry. Western Blot: 1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures. 2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at room temperature. Wash with TPBS （PBS +0.05% Tween™20）. 4. Prepare VECTASTAIN® ® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP （alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate the membrane in the reagent for 30 minutes at room temperature. Wash with TPBS. 5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™ Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB （Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity. Vector Labs offers a series of sugars that are intended for such a purpose. The lectin is diluted to a suitable working concentration in a solution containing approximay 200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min. Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
Description: Hydrophobic barrier pen for immunohistochemistry and in situ hybridization. ImmEdge™ Pen provides a heat-stable water-repellent barrier that keeps reagents localized on tissue sections. Unit Size: 2 pen set Storage Conditions: Room temperature with pen tip down and tightly capped. INSTRUCTIONS FOR USE 1. Shake vigorously. 2. Depress tip to saturate, if necessary. 3. Circumscribe tissue section with the ImmEdge™ Pen. 4. Replace pen cap. Store pen tip down. 5. Allow ImmEdge™ Pen reagent to dry before immersing slides in aqueous solutions. DISPOSAL OF REAGENTS Dispose in an approved landfill. MSDS available online at: www.vectorlabs.com For Professional Use Only. Authorized Representative / Exclusive Distributor to UK and Ireland: Vector Laboratories Ltd 3 Accent Park Bakewell Road, Orton Southgate Peterborough, PE2 6XS, United Kingdom
