ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse

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1 ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse Code No./ Code/ Code-Nr. K nd edition/ 2ème édition/ 2. Ausgabe For use with DakoCytomation TechMate and Autostainer Instruments. The kit contains reagents sufficient for 500 tests. A utiliser avec les instruments TechMate et Autostainer DakoCytomation. Le kit contient une quantité de réactifs suffisante pour 500 essais. Zur Verwendung zusammen mit den DakoCytomation TechMate - und Autostainer-Geräten. Der Kitinhalt ist für 500 Tests ausreichend. ( ) K 5007/EFG/HGW/ p. 1/47

2 Contents/ Table des matières/ Inhalt ENGLISH GENERAL INSTRUCTIONS Page/ Page/ Seite Intended Use... 5 Introduction... 5 Reagents... 5 Materials provided... 5 Precautions... 5 Storage... 6 Quality Control... 6 Interpretation of Results... 6 INSTRUCTIONS FOR DakoCytomation TechMate INSTRUMENTS Summary and Explanation... 7 Reagents... 8 A. Materials required but not provided... 8 B. Reagent preparation... 8 Specimen Collection and Preparation... 9 Procedure... 9 A. Epitope retrieval in microwave oven B. Guideline for optimal dilution of primary antibodies and proteinase K pre- treatment C. Staining protocol INSTRUCTIONS FOR DakoCytomation AUTOSTAINER INSTRUMENTS Summary and Explanation Reagents A. Materials required but not provided B. Reagent preparation...14 Specimen Collection and Preparation Procedure A. Epitope retrieval in microwave oven B. Guideline for optimal dilution of primary antibodies and proteinase K pretreatment C. Programming of the DakoCytomation Autostainer D. Staining protocol Explanation of symbols ( ) K 5007/EFG/HGW/ p. 2/47

3 FRANÇAIS CONSIGNES GENERALES Intérêt Introduction Réactifs Matériels fournis Précautions Conservation Contrôle qualité Interprétation des résultats CONSIGNES D'UTILISATION des instruments TechMate DakoCytomation Résumé et explications Réactifs A. Matériels nécessaires mais non fournis B. Préparation des réactifs Collecte et préparation des échantillons Procédure A. Restauration de l'épitope au four à micro-ondes B. Recommandations de dilution optimale pour le pré-traitement des anticorps primaires et de la protéinase K C. Protocole d'immunomarquage CONSIGNES D'UTILISATION DES INSTRUMENTS AUTOSTAINER de DakoCytomation Résumé et explications Réactifs A. Matériels nécessaires mais non fournis B. Préparation des réactifs Collecte et préparation des échantillons Procédure A. Restauration de l'épitope au four à micro-ondes B. Recommandations de dilution optimale pour le pré-traitement des anticorps primaires et de la protéinase K C. Programmation de l'autostainer de DakoCytomation D. Protocole d immunomarquage Explication des symboles ( ) K 5007/EFG/HGW/ p. 3/47

4 DEUTSCH ALLGEMEINE ANLEITUNGEN Zweckbestimmung Einleitung Reagenzien Packungsinhalt Hinweise und Vorsichtsmaßnahmen Lagerung Qualitätskontrolle Interpretation der Ergebnisse ANLEITUNGEN FÜR DakoCytomation TechMate -GERÄTE Zusammenfassung und Erläuterung Reagenzien A. Zusätzlich benötigte Reagenzien und Zubehör (außerhalb des Lieferumfangs) B. Reagenzienvorbereitung Probengewinnung und -vorbereitung Prozedur A. Epitopdemaskierung im Mikrowellengerät B. Leitlinie für die optimale Verdünnung von primären Antikörpern und für die Proteinase K-Vorbehandlung C. Färbungsprotokoll ANLEITUNGEN FÜR DakoCytomation Autostainer-GERÄTE Zusammenfassung und Erläuterung Reagenzien A. Zusätzlich benötigte Reagenzien und Zubehör (außerhalb des Lieferumfangs) B. Reagenzienvorbereitung Probengewinnung und -vorbereitung Prozedur A. Epitopdemaskierung im Mikrowellengerät B. Leitlinie für die optimale Verdünnung von primären Antikörpern und die Proteinase K-Vorbehandlung C. Programmieren des DakoCytomation Autostainer D. Färbungsprotokoll Erläuterung der Symbole ( ) K 5007/EFG/HGW/ p. 4/47

5 ENGLISH GENERAL INSTRUCTIONS Intended Use For in vitro diagnostic use. ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse, is intended for use in immunocytochemistry together with the DakoCytomation TechMate and Autostainer Instruments. The kit is employed in a two-step procedure where the first step is incubation of the tissue with an optimally diluted primary rabbit or mouse antibody, and the second step is incubation with the ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV) reagent of the kit. This reagent is a peroxidase-conjugated polymer, which also carries antibodies to rabbit and mouse immunoglobulins. The reaction is visualized by the ChemMate DAB+ Chromogen also included in the kit. Introduction ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse, is for use on the DakoCytomation TechMate and Autostainer Instruments. The instructions for the two instruments differ and are, therefore, provided in separate sections. Reagents Materials provided Bottle A Bottle B Bottle C ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV) 100 ml, ready-to-use Dextran coupled with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse immunoglobulins. In buffered solution containing stabilizing protein and preservative. ChemMate Substrate Buffer 250 ml Buffered solution containing hydrogen peroxide and preservative. ChemMate DAB+ Chromogen 5 ml, 50 x concentrated 3,3'-diaminobenzidine tetrahydrochloride in organic solvent. The colour of this reagent may vary from strong violet to colourless without having any influence on the performance of the kit. Precautions 1. For professional users. 2. Bottle A, ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV), contains material of animal origin and it cannot be excluded that trace amounts of human material could be present due to manufacturing procedures. As with any product derived from biological sources, proper handling should be used. ( ) K 5007/EFG/HGW/ p. 5/47

6 3. Do not expose Bottle C, ChemMate DAB+ Chromogen or the Substrate Working Solution (CHROM) to strong light. 4. The reagents loaded on the TechMate Instruments should not be reused. 5. Bottle C, ChemMate DAB+ Chromogen contains 1.5% 3,3 -diaminobenzidine tetrahydrochloride, and is labelled: Harmful. R40 Limited evidence of a carcinogenic effect. R43 May cause sensitisation by skin contact. R68 Possible risk of irreversible effects. S35 This material and its container must be disposed of in a safe way. S36/37 Wear suitable protective clothing and gloves. As a main rule, persons under 18 years of age are not allowed to work with this product. Users must be carefully instructed in the proper working procedure, the dangerous properties of the product and the necessary safety instructions. Please refer to the Material Safety Data Sheet (MSDS) for additional information. Storage Store the kit at 2-8 C. Do not use after expiration date stamped on the kit. Do not interchange kit components from different lots. If reagents are stored under any conditions other than those specified, the conditions must be verified by the user. The prepared Substrate Working Solution (CHROM) should be stored at 2-8 C and used within 5 days. If unexpected staining is observed which cannot be explained by variations in laboratory procedures and a problem with the product is suspected, contact our Technical Services. Quality Control Each staining run should include a known positive control specimen to ascertain a proper performance of all the applied reagents. If the positive control specimen fails to demonstrate positive staining, labelling of test specimens should be considered invalid. A negative control reagent should be used with each specimen to identify any non-specific staining. If non-specific staining cannot be clearly differentiated from the specific staining, the labelling of the test specimen should be considered invalid. Interpretation of Results The diaminobenzidine-containing Substrate Working Solution gives a brown colour at the site of the target antigen recognized by the primary antibody. The brown colour should be present on the positive control specimen at the expected localization of the target antigen. If non-specific staining is present, this will be recognized as a rather diffuse, brown staining on the slides treated with the negative control reagent. Nuclei will be stained blue by the hematoxylin counterstain. ( ) K 5007/EFG/HGW/ p. 6/47

7 INSTRUCTIONS FOR DakoCytomation TechMate INSTRUMENTS Summary and Explanation ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse, has been designed to give optimal staining on the TechMate Instruments when using the protocols outlined in the instruction below. The automatic immunostaining on the TechMate Instruments uses capillary action to draw up reagents from Reagent Trays, code No. S 2039, to cover specimens mounted on special Capillary Gap Microscope Slides. After a predetermined incubation time, reagents are removed from specimens by absorption into Absorbent Pads, code No. S 2027 for TechMate 500/500 Plus/1000, and code No. S 2043 for TechMate Horizon. Prior to staining on the TechMate Instruments, routinely fixed, paraffin-embedded tissue sections should be subjected to epitope retrieval in microwave oven using ChemMate Target Retrieval Solution, code No. S 2031, diluted 1:10. Please note that some primary antibodies require proteinase K pre-treatment of tissue for optimal staining. Endogenous peroxidase should be blocked with the Peroxidase-Blocking Solution, code No. S Owing to an effective washing procedure and the presence of carrier proteins in the DakoCytomation reagents, extra blocking steps to reduce non-specific background staining are unnecessary. The ChemMate Buffer Kit, code No. K 5006, contains Wash Buffers 1, 2, 3 and Water Wash. Wash Buffer 1 is used during the first part of the procedure. The formulation of this buffer contributes to the blocking of non-specific binding of the primary antibody and the ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV) detection reagent to the tissue sections. Wash Buffers 2 and 3 are identical and are used for the remaining part of the procedure. As Wash Buffers 2 and 3 do not contain sodium azide, they are used especially in conjunction with washing after incubation with the ENV detection reagent. Water Wash is used for washing steps at the end of the staining protocols. Primary antibodies are not provided with the kit. We recommend the use of concentrated DakoCytomation primary antibodies. Please see the guideline for optimal dilutions in Table 2. The ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV) detection reagent in the kit consists of a dextran backbone to which a large number of peroxidase (HRP) molecules and secondary antibody molecules have been coupled. A unique chemistry is used for the coupling reaction, which permits the binding of up to 100 HRP molecules and up to 20 antibody molecules per backbone. The secondary antibody coupled to the dextran backbone has been raised in goats. It reacts equally well with rabbit and mouse immunoglobulins, thus only one detection reagent is required for rabbit and mouse primary antibodies. The ENV detection reagent is provided ready-to-use in a dropper bottle. The substrate system consists of two components, ChemMate DAB+ Chromogen, a concentrated diaminobenzidine (DAB) solution, and the ChemMate Substrate Buffer containing hydrogen peroxide. Before use, the DAB+ Chromogen must be diluted in the Substrate Buffer. The substrate system produces a brown colour at the site of the target antigen. ChemMate Hematoxylin, code No. S 2020, is recommended for counterstaining as it provides a clear blue, nuclear staining. The stained tissue sections may be mounted with either aqueous or organic-solvent-based mounting medium. ( ) K 5007/EFG/HGW/ p. 7/47

8 Reagents A. Materials required but not provided DakoCytomation TechMate Instrument ChemMate Capillary Gap Microscope Slides, code Nos. S 2024 (75 µm), S 2025 (100 µm), S 2026 (155 µm) ChemMate Buffer Kit, code No. K 5006 ChemMate Peroxidase-Blocking Solution, code No. S 2023 ChemMate Proteinase K, code No. S 2019 (if necessary) ChemMate Proteinase K Diluent, code No. S 2032 (if necessary) Primary rabbit or mouse antibodies from DakoCytomation or other source Suitable negative control reagent for primary antibody ChemMate Antibody Diluent, code No. S 2022 (for dilution of concentrated antibodies) ChemMate Hematoxylin, code No. S 2020 ChemMate Target Retrieval Solution, Concentrated x 10, code No. S 2031 ChemMate Incubation Container, code No. S 2030 ChemMate Slide Holder, code No. S 2029 ChemMate Absorbent Pads, code No. S 2027 for TechMate 500/500 Plus/1000, or ChemMate Absorbent Pads, code No. S 2043 for TechMate Horizon ChemMate Reagent Trays, code No. S 2039 General laboratory reagents for dewaxing paraffin-embedded tissue sections Mounting medium (aqueous or organic-solvent-based) and coverslips B. Reagent preparation Substrate Working Solution (CHROM) The DAB-containing Substrate Working Solution (CHROM) is prepared by mixing thoroughly 50 parts of ChemMate Substrate Buffer (Bottle B) with 1 part of ChemMate DAB+ Chromogen (Bottle C). Prepare only the volume required for the number of slides that shall be stained. Please see Table 1 below. One well corresponds to two slides that have been paired. Use CHROM within 5 days (store in darkness at 2-8 C). Table 1. Number of wells (each containing approx. 750 µl) (1 tray) 20 (2 trays) 30 (3 trays) ChemMate Substrate Buffer 750 µl 4 ml 8 ml 16 ml 24 ml ChemMate DAB+ Chromogen 15 µl 80 µl 160 µl 320 µl 480 µl For the preparation of reagents not provided with the kit, please see the instructions included with the individual reagent. ( ) K 5007/EFG/HGW/ p. 8/47

9 Specimen Collection and Preparation The specimens may be routinely fixed, paraffin-embedded tissue sections and frozen tissue sections. A variety of fixatives may be used for specimen preservation. Specimens should not be fixed in neutral buffered formalin for more than 24 hours and preferably for a shorter time. The optimal thickness of paraffin-embedded sections is approximately 4 µm. The optimal thickness of frozen sections is 4-6 µm. All specimens should be mounted on ChemMate Capillary Gap Microscope Slides and placed in the slide holder "face to face" in pairs. As the slides have already been coated with adhesive, the user is advised not to treat the slides further, as this may have a detrimental effect. Three types of Capillary Gap Microscope Slides are available: 1. Slides with grey, 75 µm thick painted areas. These are for paraffin-embedded tissue sections and should be paired with similar slides. 2. Slides with blue, 100 µm thick painted areas. These are for frozen sections and smears and should be paired with similar slides. 3. Slides with yellow, 155 µm thick painted areas. These slides should not be mounted with tissue sections. They are for use with tissue sections which have been mounted on ordinary, non-capillary gap slides. Simply pair the ordinary slide with the yellow slide to create a proper capillary gap. NOTE: Ordinary slides must have a width of no more than 25 mm to be able to fit into the slide holder. Specimens should be mounted on that part of the slide which is farthest from the painted label end of the slide. It is important that specimens be placed on the same side of the slide that holds the painted label area. As the tissue sections are placed "face to face" during the staining procedure, the sections should be mounted on the slides as flat and wrinkle-free as possible. Too many wrinkles or clumps will inhibit the capillary action and may cause inconsistent staining results. Paraffin sections should be mounted from a pre-heated water bath containing distilled or deionized water. The water bath should contain no additives (such as gelatin, poly-l-lysine etc.). Sections should be dried by heating, generally at a temperature not above 60 C for a minimum of 60 minutes (e.g. overnight). To ensure proper adherence of sections to slides it is important to drain the water from beneath the sections prior to the oven drying process. Procedure Before running protocols on the DakoCytomation TechMate Instruments, please read carefully the Operator's Manual for the dedicated TechMate Instrument. Prior to starting the TechMate Instrument, slides must be organized for the staining run: Routinely fixed, paraffin-embedded tissue sections: After deparaffinization and hydration to buffer (water), tissue sections should be subjected to epitope retrieval in microwave oven (see section A below). A few epitopes do not tolerate the treatment in microwave oven. Please refer to the instructions for the individual DakoCytomation primary antibody. In addition to being heated in a microwave oven, a few epitopes require a short enzyme digestion on the TechMate Instrument. Please refer to Table 2 and to the instructions for ChemMate Proteinase K, code No. S Those specimens that require enzyme digestion should be grouped together. If no specimens require enzyme digestion, use protocol ENVP for all slides (this protocol is performed in approximately 2 ¼ hours). ( ) K 5007/EFG/HGW/ p. 9/47

10 If some specimens require enzyme digestion, use protocol ENVPE for all slides (this protocol is performed in approximately 2 ¼ hours). For those specimens that do not require enzyme digestion, simply replace the proteolytic enzyme in the well with Proteinase K Diluent, code No. S 2032, or Washing Buffer 2 or 3 from ChemMate Buffer Kit, code No. K A. Epitope retrieval in microwave oven It is recommended to follow carefully the instructions below in order to ascertain reproducible epitope retrieval. To standardize the treatment, it is recommended to load the slide holder fully with slides, even if only one slide may hold tissue; this ensures an identical heating of sections in every run. At the termination of the epitope retrieval step, slides must be left in the buffer for at least 20 minutes at room temperature. During this cooling, the TechMate Instrument can be prepared for the staining run. Microwave oven procedure 1. Place the deparaffinized tissue sections in a ChemMate Slide Holder, code No. S Transfer the ChemMate Slide Holder to a ChemMate Incubation Container, code No. S 2030, filled with 200 ml of ChemMate Target Retrieval Solution, code No. S 2031, diluted 1: Place the Container in the microwave oven with the lid at an angle (e.g. diagonally). Use an 800 watt microwave oven on high setting. Microwave for 5 minutes after the buffer has come to a boil. Add 50 ml of deionized water to replace the evaporated water. Microwave for another 5 minutes. Remove the Container from the oven and let it cool at room temperature for at least 20 minutes. 3. Exchange the buffer with deionized water and leave for a short time. Now the slides are ready to be loaded into the TechMate Slide Holder. NOTE: Boiling of the buffer is normal and desirable. Not all 800 watt microwave ovens are similar in heat transfer. Therefore, to secure a local standardization, the microwave oven should be tested with slides without tissue the first time to find the total microwaving time necessary for obtaining 5 minutes of actual boiling. B. Guideline for optimal dilution of primary antibodies and proteinase K pretreatment Table 2 provides a dilution guideline for DakoCytomation concentrated primary antibodies used with the ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse, and applied on formalin-fixed, paraffin-embedded sections. The primary antibodies should be diluted in ChemMate Antibody Diluent, code No. S ( ) K 5007/EFG/HGW/ p. 10/47

11 Table 2. Guideline for optimal dilution of primary antibodies and proteinase K pre- treatment Code No. Primary Antibody Proteinase K Pre-treatment Optimal Dilution for TechMate Instruments A 0010 Rabbit Anti-Human Synaptophysin YES 1:50-1:100 A 0082 Rabbit Anti-Human Von Willebrand Factor YES 1:250-1:500 A 0098 Rabbit Anti-Human Progesterone Receptor - 1:5-1:10 A 0191 Rabbit Anti-Human Kappa Light Chains YES 1:2000-1:4000 A 0193 Rabbit Anti-Human Lambda Light Chains YES 1:500-1:1000 A 0251 Rabbit Anti-Human Thyroglobulin - 1:400-1:1200 Z 0311 Rabbit Anti- S100 YES 1:250-1:500 Z 0334 Rabbit Anti-Glial Fibrillary Acidic Protein - 1:400-1:1200 A 0430 Rabbit Anti-Human Chromogranin A - 1:1000-1:2000 A 0452 Rabbit Anti-Human CD3, T Cell YES 1:50-1:100 A 0562 Rabbit Anti-Human Prostate-Specific Antigen - 1:1000-1:4000 A 0576 Rabbit Anti-Human Calcitonin - 1:100-1:200 M 0613 Mouse Anti-Human Epithelial Membrane Antigen, Clone E29-1:40-1:80 M 0630 Mouse Anti-Human Cytokeratin, High Molecular Weight, Clone 34βE12 YES 1:25-1:50 M 0631 Mouse Anti-Human Cytokeratin 8, Low Molecular Weight, Clone 35βH11-1:20-1:40 M 0634 Mouse Anti Human Melanosome, Clone HMB-45-1:50-1:100 M 0635 Mouse Anti-Human Muscle Actin, Clone HHF35-1:20-1:40 M 0701 Mouse Anti-Human CD45, Leucocyte Common Antigen, Clones 2B11 + PD7/26-1:50-1:100 M 0725 Mouse Anti-Vimentin, Clone V9-1:10-1:20 M 0742 Mouse Anti-Human CD45R0, Clone UCHL1-1:50-1:100 M 0751 Mouse Anti-Human CD30, Clone Ber-H2 YES 1:3-1:6 M 0755 Mouse Anti-Human CD20cy, Clone L26-1:50-1:200 M 0760 Mouse Anti-Human Desmin, Clone D33-1:50-1:100 M 0785 Mouse Anti-Human Collagen IV, Clone CIV 22 YES 1:5-1:10 M 0821 Mouse Anti-Human Cytokeratin, Clone MNF116 YES 1:50-1:100 M 0851 Mouse Anti-Human Smooth Muscle Actin, Clone 1A4-1:50-1:100 M 0873 Mouse Anti-Human Neuron-Specific Enolase, Clone BBS/NC/VI-H14-1:200-1:800 M 0876 Mouse Anti-Human CD68, Clone PG-M1-1:50-1:100 M 0879 Mouse Anti-Proliferating Cell Nuclear Antigen, Clone PC10-1:1000-1:2000 M 0887 Mouse Anti-Human BCL2 Oncoprotein, Clone 124-1:2-1:6 M 7001 Mouse Anti-Human p53 Protein, Clone DO-7-1:25-1:100 M 7047 Mouse Anti-Human Estrogen Receptor α, Clone 1D5-1:5-1:10 M 7050 Mouse Anti-Human CD79α, Clone JCB117-1:50-1:100 M 7072 Mouse Anti-Human Carcinoembryonic Antigen, Clone II-7-1:10-1:20 C. Staining protocol The protocols ENVP and ENVPE, and templates for reagent positions on the instrument can be viewed on the TechMate screen before and during the run. The protocols list number of steps, step name and incubation times for the individual steps. The templates give the positions of the reagents and absorbent pads on the TechMate Instrument. It is recommended to fill in an Antibody Positioning Sheet for slide pairs which should be treated individually with reagents, e.g. primary antibodies, from the 10-well trays. Please refer to the Operator's Manual. ( ) K 5007/EFG/HGW/ p. 11/47

12 1. Load reagents into the 10-well trays on the TechMate Instrument according to Reagent Template and Antibody Positioning Sheet. Take care to load proper reagents and proper volumes. Pay particular attention to the loading of primary antibodies. 2. Prior to loading slides into the slide holder it is recommended to pre-wet the slides with Wash Buffer 2 from the ChemMate Buffer Kit, code No. K This can be done conveniently by leaving the slides in pairs, and organized according to the Antibody Positioning Sheet, for a few minutes in a 10-well tray containing Wash Buffer 2. This prewetting will ensure adequate fluid flow in the capillary gap. 3. After pre-wetting, place the slide pairs in the TechMate Slide Holder according to the Antibody Positioning Sheet. Please make sure that the slides are in alignment. 4. Just before the run, place the TechMate Slide Holder on Pad2 in the TechMate Instrument to drain Wash Buffer 2 from the capillary gap. Observe carefully the draining of slides in order to control that all slides have been correctly paired and that the alignment of slide pairs has been done properly. 5. Place the TechMate Slide Holder in the Wash Buffer (e.g. Wash Buffer 1 or Wash Buffer 2) used in the first step of the protocol to be run, in order to fill the capillary gaps with Wash Buffer. 6. Place the TechMate Slide Holder in HOME position before starting the run. Please refer to Chapter 5 "RUNNING A SINGLE PROTOCOL" in the Operator's Manual. NOTE: The final steps in the protocols are washing in Water Wash (from ChemMate Buffer Kit, code No. K 5006), designated H2O on the template and in the protocol. If the user prefers to prepare the specimens for mounting in aqueous mounting medium, the HOME position can be loaded with water after initiation of the run. On TechMate Horizon, the H2O position is equal to the HOME position. The volumes to be loaded into the trays for the individual reagents are given below. These volumes fit with the protocols used for this kit. Wash Buffers 1, 2 or 3 (BUF1, 2, 3) Water Wash (H2O) Peroxidase-Blocking Solution (HP-BK) Substrate Working Solution (CHROM), prepared from this kit Diluted Proteinase K (ENZ) Hematoxylin (HEMA) Primary antibody (AB1) EnVision /HRP (ENV), Bottle A in this kit 20 ml per 10-well tray 20 ml per 10-well tray 10 ml per 10-well tray 750 µl per individual well 500 µl per individual well 750 µl per individual well 8 drops (320 µl) per individual well 8 drops (320 µl) per individual well ( ) K 5007/EFG/HGW/ p. 12/47

13 INSTRUCTIONS FOR DakoCytomation AUTOSTAINER INSTRUMENTS Summary and Explanation ChemMate DAKO Envision Detection Kit, Peroxidase/DAB, Rabbit/Mouse, has been adapted for use on the DakoCytomation Autostainer Instruments according to the template outlined below. Prior to staining on the Autostainer Instruments, some routinely fixed, paraffin-embedded tissue sections should be subjected to epitope retrieval in microwave oven using the Target Retrieval Solution specified in Table 3 or the instructions for the primary antibody. Please note that some primary antibodies require additional proteinase K pre-treatment of tissue for optimal staining. Endogenous peroxidase should be blocked with Peroxidase-Blocking Solution, code No. S Owing to an effective washing procedure and the presence of carrier proteins in the DakoCytomation reagents, extra blocking steps to reduce non-specific background staining is unnecessary. The DakoCytomation Wash Buffer, Concentrated x 10, for Immunocytochemistry, code No. S 3006, is recommended. Primary antibodies are not provided with the kit. We recommend the use of concentrated DakoCytomation Antibodies. Please see the guideline for optimal dilution in Table 3. The ChemMate DAKO EnVision /HRP, Rabbit/Mouse (ENV) detection reagent in the kit consists of a dextran backbone to which a large number of peroxidase (HRP) molecules and secondary antibody molecules have been coupled. A unique chemistry is used for the coupling reaction, which permits the binding of up to 100 HRP molecules and up to 20 antibody molecules per backbone. The secondary antibody coupled to the dextran backbone has been raised in goats. It reacts equally well with rabbit and mouse immunoglobulins, thus only one detection reagent is required for rabbit and mouse primary antibodies. The ENV detection reagent is provided in a dropper bottle and has to be poured into an Autostainer Reagent Vial, code No. S 3425, before use. The substrate system consists of two components, ChemMate DAB+ Chromogen, a concentrated diaminobenzidine (DAB) solution, and the ChemMate Substrate Buffer containing hydrogen peroxide. Before use, the DAB+ Chromogen must be diluted in the Substrate Buffer. The substrate system produces a brown colour at the site of the target antigen. The mixed chromogen solution should be poured into an Autostainer Reagent Vial, code No. S 3425, before use. DakoCytomation Hematoxylin, code No. S 3301, is recommended for counterstaining on the Autostainer Instruments as it provides a clear blue, nuclear staining. The stained tissue sections may be mounted with either aqueous or organic-solvent-based mounting medium. Reagents A. Materials required but not provided DakoCytomation Autostainer Instrument DakoCytomation Wash Buffer, Concentrated x 10, for Immunocytochemistry, code No. S 3006 ChemMate Peroxidase-Blocking Solution, code No. S 2023 ChemMate Proteinase K, code No. S 2019 (if necessary) ChemMate Proteinase K Diluent, code No. S 2032 (if necessary) Primary rabbit or mouse antibodies from DakoCytomation or other source Suitable negative control reagent for primary antibody ( ) K 5007/EFG/HGW/ p. 13/47

14 ChemMate Antibody Diluent, code. No. S 2022 (for dilution of concentrated antibodies) DakoCytomation Hematoxylin, for use with the Autostainer Instrument, code No. S 3301 DakoCytomation Target Retrieval Solution, code No. S 1699 or S 1700, DakoCytomation Target Retrieval Solution, High ph, code No. S3307 or S3308, or ChemMate Target Retrieval Solution, Concentrated x 10, code No S 2031 ChemMate Incubation Container, code No. S 2030 ChemMate Slide Holder, code No. S 2029 DakoCytomation Autostainer Reagent Vials (100 vials), code No. S 3425 Microscope slides General laboratory reagents for dewaxing paraffin-embedded tissue sections Mounting medium (aqueous or organic-solvent-based) and coverslips B. Reagent preparation Substrate Working Solution (CHROM) The DAB-containing Substrate Working Solution (CHROM) is prepared by mixing thoroughly 20 µl ChemMate DAB+ Chromogen (Bottle C) and 1 ml ChemMate Substrate Buffer (Bottle B). Prepare only the volume required for the number of slides that shall be stained. Use CHROM within 5 days (store in darkness at 2-8 C). For preparation of reagents not provided with the kit, please see the instructions included with the individual reagent. Specimen Collection and Preparation The specimens may be routinely fixed, paraffin embedded tissue sections and frozen tissue sections. A variety of fixatives may be used for specimen preservation. Specimens should not be fixed in neutral buffered formalin for more than 24 hours and preferably for a shorter time. The optimal thickness of paraffin-embedded sections is approximately 4 µm. The optimal thickness of frozen sections is 4-6 µm. The specimens should be mounted on microscope slides. The sections should be mounted on the slides as flat and wrinkle-free as possible. Too many wrinkles will have an impact on the staining results. NOTE: The microscope slides must have a width of no more than 26 mm to be able to fit into the slide holder. Paraffin sections should be mounted from a pre-heated water bath containing distilled or deionized water. The water bath should contain no additives (such as gelatin, poly-l-lysine etc.). Sections should be dried by heating, generally at a temperature not above 60 C for a minimum of 60 minutes (e.g. overnight). To ensure proper adherence of sections to slides it is important to drain the water from beneath the sections prior to the oven drying process. Procedure Before staining according to the template on the DakoCytomation Autostainer instruments, please read carefully the Operators Manual for the dedicated Autostainer Instrument. Prior to starting the Autostainer Instrument, slides and reagents must be placed according to the Slide Layout Map and the Reagent Layout Map. Routinely fixed, paraffin-embedded tissue sections After deparaffinization and hydration to buffer (water), tissue sections should be subjected to epitope retrieval in microwave oven (see section A below). A few epitopes do not tolerate the treatment in microwave oven. Please refer to the instructions for the individual DakoCytomation primary antibody. ( ) K 5007/EFG/HGW/ p. 14/47

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