Power-StainTM 2
Contents
1. Positive Negative Control Control Tissue Tissue Positive Negative Control Control Tissue Tissue Test Tissue Reagent Control Analysis of Result Specimen contains the antigen Specimen does not contain the antigen Reagent Test Control Tissue Analysis of Result No staining Weak staining High background staining Possible causes and suggested action for No staining on any slide 1 2 3 4 Reagents not used in correct order gt Repeat procedure following Staining Protocol Instructions Substrate Chromogen reagent s not prepared properly gt Prepare a fresh Substrate Chromogen solution following product s instructions 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 Wrong Pretreatment gt Repeat procedure using correct pretreatment Possible cause and suggested action for Weak staining on all slides 30959 Rev 01 1 4 Substrate Chromogen reagent s has expired gt Prepare a fresh Substrate Chromogen solution following product s instructions Incubation times were not long enough gt Repeat procedure following Staining Protocol Instructions using incubation times specified Specimen retained too much liquid aft2. 6 Genemed Biotechnologies Inc Power Stain 1 0 Double Stain Kit Poly HRP for Mouse Poly AP for Rabbit Cat No 52 0023 Intended Use Summary And Explanation Reagents Supplied Storage Materials Required But Not Supplied Precautions 30959 Rev 01 Genemed Biotechnologies Inc ed 458 Carlton Ct South San Francisco CA 94080 U S A Tel 650 952 0110 Fax 650 952 1060 Quantity 15 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 proteins antigens 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 and Poly AP alkaline phoshatase conjugates to locate where the mouse or rabbit primary antibody is bound to the target antigens The complexes formed between Poly HRP conjugate with mouse primary antibody and Poly AP conjugate with rabbit primary antibody are observed through the use of two substrate chromogen solutions which when added sequentially result in two colored p
3. ed antigen Negative Tissue A tissue that does not contain the desired antigen Reagent Control A slide to be treated with a homologous non immune immunoglobulin Cat No 60 0045 7 or Cat No 60 0060 7 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 Cocktail mouse and rabbit to optimum concentration Dilute with Primary Antibody Diluent b Add 2 drops 100 uL or as much as needed of Primary Antibody Cocktail 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 Poly AP Conjugate Incubation 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 chromogen solutions according to product s specification sheet Chromogens recommended DAB substrate solution for HRP Conjugate and Fast Red substrate solution for AP Conjugate b Add 100 uL of substrate for HRP conjugate e g DAB
4. er rinsing steps gt Tap off excess liquid and carefully wipe around specimen after rinsing steps Enzyme Conjugate exposed to sodium azide gt Use buffer without sodium azide or check if reagent is contaminated with sodium azide during use or aliquot pipetting 5 Primary antibody dilution was incorrect gt Repeat procedure following Staining Protocol Instructions using incubation times specified gt Repeat procedure using correct dilution for primary antibody 6 Insufficient Pretreatment gt Repeat procedure using correct pretreatment Page 3 of 4 Genemed Biotechnologies Inc SEn GmbH CE 458 Carlton Ct chiffgraben 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 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 t
5. ffer 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 Sodium Azide inhibits peroxidase activity Use caution when handling Poly HRP and Poly AP Conjugate to prevent any contamination with other reagents containing sodium azide 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 MDSS GmbH C Schiffgraben 41 30175 Hannover Germany Biotechnologies Inc Gat oF Genemed Procedural Notes Preliminary Preparation Of Slides Control Slides Staining Protocol 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 the reagent provided in the kit Reagent 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 reagent is 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 desir
6. han 10 slides at a time before adding next solution 6 Wrong Pretreatment gt Repeat procedure using correct pretreatment Symbols LOT y z In Vitro Diagnostic Use Catalog No Batch No Temperature Range Use By 30959 Rev 01 Page 4 of 4 Genemed Biotechnologies Inc SEn Saye CE 458 Carlton Ct chiffgraben eal South San Francisco CA 94080 U S A 30175 Hannover Tel 650 952 0110 Germany Fax 650 952 1060
7. or as much as needed to completely cover each tissue Refer to product s specification sheet for incubation time c Rinse 3 times with Wash Buffer for 2 minutes each to remove excess substrate solution d Add 100 uL of substrate for AP conjugate e g Fast Red or as much as needed to completely cover each tissue Refer to product s specification sheet for incubation time e Rinse slides with tap water to remove excess substrate solution f Proceed with normal counterstaining and mounting protocol Step 5 Counterstaining a Counterstain according to manufacturer s instruction Note Some Chromogens e g Fast Red forms an end product which is soluble in organic compounds Therefore it is necessary to use a nonalcoholic counterstain such as Mayer s or Gill s Hamatoxylin and an aqueous based mounting medium Do not use mounting media 30959 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 Interpretation Of enemed Biotechnologies Inc containing organic solvent Step 6 Mounting Staining Results Troubleshooting a Mount and coverslip the specimen with appropriate mounting Step 1 Review Positive and Negative Controls Do not proceed to next step if the staining intensity does not meet requirements Step 2 Score the tested specimens
8. recipitates at the antigen locations The staining location and pattern is easily observable by light microscopy One bottle of ready to use Poly HRP Conjugate for Mouse Poly AP for Rabbit in an enzyme conjugate buffer containing stabilizing proteins and anti microbial agents Store at 2 8 C Do not freeze All performance claims are void after the kit expiration date Mouse and Rabbit Primary Antibodies Genemed offers prediluted and concentrate Primary Antibodies Primary Antibody Diluent Cat No 10 0001 Reagent Control Non immune Mouse IgG Cat No 60 0045 7 and Non Immune Rabbit IgG Cat No 60 0060 7 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 Substrate Chromogen Solution for HRP Conjugate e g Cat No 10 0006 DAB Substrate Kit Cat No 10 0048 Sensitive DAB Substrate Kit Cat No 10 0005 10 0047 AEC Substrate Single Solution Substrate Chromogen Solution for AP Conjugate e g Cat No 10 0008 Liquid Fast Red Substrate Kit 10 0007 BCIP NBT Substrate Kit 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 Bu
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