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Power-StainTM 2

1. Gat oF Genemed Biotechnologies Inc Power Stain 1 0 Poly HRP DAB Kit for Mouse Rabbit Cat No 52 0017 54 0017 Intended Use Summary And Explanation Reagents Supplied Storage Materials Required But Not Supplied Precautions 30408 Rev 01 Genemed Biotechnologies Inc east 458 Carlton Ct South San Francisco CA 94080 U S A Tel 650 952 0110 Fax 650 952 1060 Quantity 15 mL 100 mL For In Vitro Diagnostic Use This kit is intended for use with Mouse and Rabbit Primary Antibodies and other ancillary reagents supplied by user for qualitative detection of targeted protein antigen using immunohistochemistry IHC methodology by light microscopy on routine formalin fixed paraffin embedded FFPE tissue section Interpretation of any positive or negative staining shall be supported by implementation of a proper control and must be made within the context of the patient s clinical history and other diagnostic test by a qualified pathologist This kit is a non biotin system and utilizes a Poly HRP horseradish peroxidase conjugate to locate where the mouse or rabbit primary antibody is bound to the target antigen The complex formed between Poly HRP conjugate and the mouse or rabbit primary antibody is observed through the use of a substrate chromogen solution which when added results in a colored precipitate at the antigen location The staining location and pattern is easily observab
2. Proper handling of this product as with any product derived from biological sources should be used according to local and applicable regulations Page 1 of 4 CE MDSS GmbH Schiffgraben 41 30175 Hannover Germany Gat oF Genemed Procedural Notes Preliminary Preparation Of Slides Control Slides Staining Protocol Biotechnologies Inc Risk Statements DAB Chromogen R40 Limited evidence of carcinogenic effect R43 May cause sensitization by skin contact R68 Possible risk of irreversible effects The directions accompanying this kit provide step by step instructions for optimal staining Any change in procedure or incubation times may give erroneous staining results For optimal results do not substitute reagents provided in the kit Reagent A shall be equilibrated to room temperature readily before usage All incubations should be performed at room temperature in a humid environment Do not allow the tissue section to dry out at any point in the staining procedure The reagents are for single use Routine de paraffinization and rehydration of tissue section Antigen retrieval as required by the primary antibody Three types of control slides are necessary for proper interpretation Positive Tissue Control A tissue containing the desired antigen Negative Tissue A tissue that does not contain the desired antigen Reagent Control A slide to be treated with a homologous non immune immunoglobu
3. staining e causes and suggested action for No staining on any slide 1 Reagents not used in correct order gt Repeat procedure following Staining Protocol Instructions 2 Substrate Chromogen reagent not prepared properly gt Prepare a fresh Substrate Chromogen solution following the instructions included with the product 3 Primary antibody incubation steps were omitted or dilution was incorrect or wrong antibody was used gt Repeat procedure following Staining Protocol Instructions using incubation times specified gt Repeat procedure using correct dilution for primary antibody or correct primary antibody 4 Wrong Pretreatment gt Repeat procedure using correct pretreatment e cause and suggested action for Weak staining on all slides 1 Substrate Chromogen reagent has expired gt Prepare a fresh Substrate Chromogen solution following the instructions included with the product 2 Incubation times were not long enough gt Repeat procedure following Staining Protocol Instructions using incubation times specified 3 Specimen retained too much liquid after rinsing steps gt Tap off excess liquid and carefully wipe around specimen after rinsing steps 4 Peroxidase Enzyme Conjugate Reagent A exposed to Sodium Azide gt Use buffer without Sodium Azide or check if Reagent A is contaminated with Sodium Azide during use or aliquot pipetting 5 Primary antibody dilution was incorrect gt Repeat procedure f
4. Francisco CA 94080 U S A 30175 Hannover Tel 650 952 0110 Germany Fax 650 952 1060
5. le by light microscopy Reagent A One bottle of ready to use Poly HRP Conjugate for Mouse Rabbit in an enzyme conjugate buffer containing stabilizing proteins and anti microbial agents Reagent B1 One bottle of 2X DAB Chromogen Solution Reagent B2 One bottle of 2X DAB Buffer Solution Store at 2 8 C Do not freeze All performance claims are void after the kit expiration date Primary Antibody Genemed offers prediluted and concentrate Primary Antibodies Primary Antibody Diluent Cat No 10 0001 Reagent Control Non immune Mouse IgG Cat No 60 0045 and Non Ilmmune Rabbit IgG Cat No 60 0060 Positive and Negative Control Specimens Microscope Slides Positively Charged Xylene Ethanol Endogenous Peroxidase Blocking Solution 3 Hydrogen Peroxide Cat No 10 0056 Wash Buffer 10 mM Phosphate Buffer Saline pH 7 4 optional with 0 05 Tween 20 Hematoxylin Cat No 10 0027 10 0049 Antigen retrieval reagents e g Cat No 10 0022 Citrate Buffer pH 6 0 1X Cat No 10 0020 Citrate Buffer pH 6 0 20X Cat No 10 0021 Tris Buffer pH 9 20X Cat No 10 0023 Tris Buffer pH 9 1X Cat No 10 0046 Tris EDTA Buffer pH 9 1X Cat No 10 0037 Tris EDTA Buffer pH 9 20X Cat No 10 0024 Proteinase K Cat No 10 0025 Trypsin Cat No 10 0050 Ficin For professional users only DAB Chromogen Solution Reagent B1 is susceptible to contamination from oxidizing agents To avoid contamination do not pipette Reagent B1 directly out of the bottle
6. lin Cat No 60 0045 or Cat No 60 0060 Step 1 Endogenous Peroxidase Blocking a Submerge slides in Peroxidase Blocking Solution for 10 minutes b Wash slides with Wash Buffer to remove excess Peroxidase Blocking Solution c Tap off excess liquid and carefully wipe around tissue Step 2 Primary Antibody Incubation a Prepare Primary Antibody to optimum concentration If necessary dilute with Primary Antibody Diluent b Add 2 drops 100 uL or as much as needed of Primary Antibody to completely cover each tissue c Incubate for 30 60 minutes at room temperature d Rinse 3 times with Wash Buffer for 2 minutes each e Tap off excess liquid and carefully wipe around tissue Step 3 Poly HRP Conjugate Incubation Reagent A a Add 2 drops 100 uL or as much as needed of Enzyme Conjugate to completely cover each tissue b Incubate for 15 1 minutes c Rinse 3 times with Wash Buffer for 2 minutes each d Tap off excess liquid and carefully wipe around tissue Step 4 Substrate Chromogen a Prepare Ready To Use DAB substrate solution Add DAB Chromogen Solution Reagent B1 to DAB Buffer Solution Reagent B2 and mix the two solutions in a 1 1 ratio with the volume determined by the number of slides to stain In general 200 uL of mixed substrate solution is sufficient to cover one tissue slide Note Do not equilibrate the entire bottle of either reagent at Room Temperature Take out the necessary amount of each solutio
7. n and allow the aliquoted volumes to equilibrate at Room Temperature before mixing After mixing in a 1 1 ratio the resulting Ready To Use substrate solution should be used within 2 hours b Add substrate solution on slides and incubate for 5 10 minutes at room temperature c Rinse slides with tap water to remove excess substrate solution 30408 Rev 01 Page 2 of 4 Genemed Biotechnologies Inc MDSS GmbH 458 Carlton Ct Schiffgraben 41 nial South San Francisco CA 94080 U S A 30175 Hannover Tel 650 952 0110 Germany Fax 650 952 1060 ju Oo cy Biotechnol Step 5 Step 6 Interpretation Of Step 1 Staining Results Troubleshooting Step 2 DN Possibl Possibl 30408 Rev 01 Genemed ogies Inc d Proceed with normal counterstaining and mounting protocol Counterstaining a Counterstain with Hematoxylin according to manufacturer s instruction Mounting a Mount and coverslip the specimen with appropriate mounting Review Positive and Negative Controls Do not proceed to next step if the staining intensity does not meet requirements Score the tested specimens Positive Negative n i eagent est ome ome Carico Tice Analysis of Result Tissue Tissue p f Semen contains the antigen re ee O Specimen does not contain the antigen Negative 7 m eagent est p Control Tissue Analysis of Result Tissue No staining Positive Control Tissue High background
8. ollowing Staining Protocol Instructions using incubation times specified Page 3 of 4 Genemed Biotechnologies Inc MDSS GmbH 458 Carlton Ct Schiffgraben 41 South San Francisco CA 94080 U S A 30175 Hannover Tel 650 952 0110 Germany Fax 650 952 1060 Gat ofp Genemed Biotechnologies Inc gt Repeat procedure using correct dilution for primary antibody 6 Insufficient Pretreatment gt Repeat procedure using correct pretreatment Possible cause and suggested action for High background staining on all slides 1 Specimens contain high endogenous peroxidase activity gt Check preparation of Peroxidase Solution and verify timing of specimens submerged in solution 2 Inadequate rinsing of slides gt Use freshly prepared buffer solutions Follow rinsing instructions specified 3 De paraffinization not complete gt Use fresh xylene Check slides are de paraffinized before rehydration step 4 Over reaction of substrate gt Do not incubate substrate longer than specified in procedure 5 Specimens dry out during staining procedure gt Incubate in humid environment Wipe fewer than 10 slides at a time before adding next solution 6 Wrong Pretreatment gt Repeat procedure using correct pretreatment Symbols REF LOT yt Z Catalog No Batch No In Vitro Diagnostic Use Temperature Range Use By 30408 Rev 01 Page 4 of 4 Genemed Biotechnologies Inc SEn U aA CE 458 Carlton Ct chiffgraben eal South San

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