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HCS30 BrdU Immunohistochemistry System

BrdU Immunohistochemistry System MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
HCS30
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Overview

Replacement Information

Key Spec Table

Detection Methods
Microscopy

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HCS30-1EA
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      Description
      OverviewUseful for the detection of proliferating cells within fixed tissue samples or cell populations. Requires metabolic incorporation of BrdU (Cat. No. 203806) into sample prior to paraffin-embedding.
      Catalogue NumberHCS30
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Phosphate buffered saline (PBS)
      • Quenching solution for endogenous peroxidase
      • Alcohol and xylene
      • Coverslips
      • BrdU (i.e. Calbiochem Cat. No. 203806)
      References
      ReferencesZhang, Q. and Paria, B.C. 2006. Endocrinology 147, 2215.
      Fukuda, K ., et al. 1990. Anal. Quant. Cytol. Histol. 12, 135.
      Cattoretti, G. et al. 1993. J. Pathol. 171, 83.
      Schutte, B., et al. 1987. J. Histochem. & Cytochem. 35,1343.
      Gonchoroff , N.J., et al. 1986. J. Immunol. Meth. 93, 97.
      Ellwart, E. and Dormer, P. 1985. Cytometry 6, 513.
      Gratzner, H.G., et al. 1975. Expl. Cell Res. 95, 88.
      Product Information
      Detection methodMicroscopy
      Form50 Tests
      Kit containsTrypsin concentrate, trypsin diluent, denaturing solution, blocking solution, biotinylated sheep anti-BrdU, streptavidin-peroxidase, substrate buffer reaction mix, DAB concentrate, hematoxylin, mounting media, control slides (1 stained and 4 unstained), and a user protocol.
      Positive controlAny cell or tissue sample labeled with BrdU
      Applications
      Biological Information
      Assay time~2.5 hours plus labeling time
      Sample TypeParaffin-embedded tissues or fixed cells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 25-36/37/38-42/43-61

      Toxic if swallowed.
      Irritating to eyes, respiratory system and skin.
      May cause sensitization by inhalation and skin contact.
      May cause harm to the unborn child.
      S PhraseS: 24-26-36/37/39-45

      Avoid contact with skin.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -20°C. Avoid contact with DAB; wearing of latex or rubber gloves is recommended.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsTrypsin concentrate, trypsin diluent, denaturing solution, blocking solution, biotinylated sheep anti-BrdU, streptavidin-peroxidase, substrate buffer reaction mix, DAB concentrate, hematoxylin, mounting media, control slides (1 stained and 4 unstained), and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      HCS30-1EA 04055977228342

      Documentation

      BrdU Immunohistochemistry System SDS

      Title

      Safety Data Sheet (SDS) 

      BrdU Immunohistochemistry System Certificates of Analysis

      TitleLot Number
      HCS30

      References

      Reference overview
      Zhang, Q. and Paria, B.C. 2006. Endocrinology 147, 2215.
      Fukuda, K ., et al. 1990. Anal. Quant. Cytol. Histol. 12, 135.
      Cattoretti, G. et al. 1993. J. Pathol. 171, 83.
      Schutte, B., et al. 1987. J. Histochem. & Cytochem. 35,1343.
      Gonchoroff , N.J., et al. 1986. J. Immunol. Meth. 93, 97.
      Ellwart, E. and Dormer, P. 1985. Cytometry 6, 513.
      Gratzner, H.G., et al. 1975. Expl. Cell Res. 95, 88.

      Citations

      Title
    • Zhang Q., and Paria B.C., 2006. Endocrinology 147, 2215.
      		•
      User Protocol

      Revision09-July-2020 JSW
      Form50 Tests
      Detection methodMicroscopy
      StorageUpon arrival store the entire contents of the kit at -20°C. Avoid contact with DAB; wearing of latex or rubber gloves is recommended.
      BackgroundMany cell proliferation studies have used the incorporation of radioactive thymidine as evidence of DNA replication. As an alternative non-radioactive technique, bromodeoxyuridine (BrdU), a thymidine analog, can be incorporated into proliferating cells (S-phase) much like radioactive thymidine. The BrdU is then detected by a monoclonal anti-BrdU antibody and visualized using a highly sensitive streptavidin-biotin staining system. BrdU has proven useful for proliferative studies of normal and neoplastic tissues both in vivo and in vitro. The BrdU Immunohistochemistry System uses a biotinylated monoclonal anti-BrdU, eliminating the need for a species-specific antibody. As a result, BrdU labeling can be performed in rats and mice without problems related to cross-reactivity or non-specific background staining. Staining is visualized using streptavidin-peroxidase and diaminobendizine (DAB), which will stain BrdU-incorporated nuclei a dark brown. This kit contains five BrdU positive control slides of tissue containing BrdU-labeled DNA (1 stained and 4 unstained slides).
      Materials provided• Trypsin Concentrate (Reagent 1A) (Kit Component No. JA7570): 1 bottle
      • Trypsin Diluent (Reagent 1B) (Kit Component No. JA7571): 1 bottle
      • Denaturing Solution (Reagent 2) (Kit Component No. JA7572): 1 bottle
      • Blocking Solution (Reagent 3) (Kit Component No. JA7573): 1 bottle
      • Biotinylated Sheep Anti-BrdU (Reagent 4) (Kit Component No. JA7574): 1 bottle
      • Streptavidin-Peroxidase (Reagent 5) (Kit Component No. JA7575): 1 bottle
      • Substrate Buffer Reaction Mix (Reagent 6A) (Kit Component No. JA7576): 1 bottle
      • DAB Concentrate (20X) (Reagent 6B) (Kit Component No. JA7577): 1 vial
      • Hematoxylin (Reagent 7) (Kit Component No. JA7578: 1 vial
      • Mounting Media (Reagent 8) (Kit Component No. JA7579): 1 bottle
      • Control Slide Set (Kit Component No. JA7580): 1 Stained and 4 Unstained
      Materials Required but not provided• Phosphate buffered saline (PBS)
      • Quenching solution for endogenous peroxidase
      • Alcohol and xylene
      • Coverslips
      • BrdU (i.e. Calbiochem Cat. No. 203806)
      Reagent preparationA. Paraffin-Embedded Tissues 1. PEROXIDASE QUENCHING SOLUTION: Add one part 30% H2O2 to 9 parts absolute methanol. Mix well. • Submerge slides in Quenching solution. After incubation, rinse with PBS for 2 min. (Inclubation time: 10 min) 2. *TRYPSIN: Add 1 drop of reagent 1A to 3 drops of reagent 1B. Mix well. • FOR FORMALIN FIXED TISSUE ONLY (Not necessary for alcohol fixed tissues). Add 2 or more drops to each section. Incubate in moist chamber at room temperature. Rinse in distilled water for 2 min. (Inclubation time: 3-10 min) 3. Alternate epitope exposure methods • see Cattoretti et al. 1993. J. Pathol 171, 83. (Inclubation time: 10 min) 4. DENATURING SOLUTION:** Reagent 2 (Ready-to-use) • Apply 2 drops or more to each section. Incubate at room temperature. Rinse with PBS for 2 min. (Inclubation time: 30 min) 5. BLOCKING SOLUTION: Reagent 3 (Ready-to-use). • Apply 2 drops or 100 µl to each section. Incubate room temperature. Drain or blot solution. DO NOT RINSE. (Inclubation time: 10 min) 6. BIOTINYLATED SHEEP ANTI-BrdU: Reagent 4 (Ready-to-use). • Apply 2 drops or 100 µl to each section. Incubate at room temperature. Rinse with PBS for 2 min. Drain or blot solution. (Inclubation time: 60 min) 7. STREPTAVIDIN-PEROXIDASE: Reagent 5 (Ready-to-use). • Apply 2 drops or 100 µl to each section. Incubate at room temperature. Rinse with PBS (2 min, 3 times). (Inclubation time: 10 min) 8. DAB MIXTURE: Add 1 drop (40 µl) of concentrated DAB substrate to 1 ml of reaction mix 6A. Mix well. Protect from light and use within 1 h. • Apply 2 or more drops of DAB MIXTURE to each section. Incubate. Rinse well with distilled water.If running less than 10 slides it may be helpful to remove the dropper bottle tips and pipet 4 µl of DAB concentrate for every 100 µl of substrate reaction mix. (Inclubation time: 2-5 min) 9. HEMATOXYLIN: Reagent 7 (Ready-to-use). • Counterstain the slides with 2 drops of HEMATOXYLIN. Wash slides in tap water. Put slides into PBS until sections turn blue (~30 s). Rinse in distilled water. (Inclubation time: 1-5 min) 10. MOUNTING MEDIA: Reagent 8 (Ready-to-use). • Dehydrate slides in 90% ethanol (10 s, 2 times), 100% ethanol (10 s, 2 times) and xylene (10 s, 2 times). Add 2 drops of Mounting media and coverslip. *Depending upon the fixative condition, the concentration of Trypsin may be varied within a range of 0.05% to 0.25% (dilute 1A with 1B from 1:10 to 1:2). B. Cultured Cells and Cell Suspensions 1. DENATURING SOLUTION:** Reagent 2 (ready to use). • Incubate with DENATURING SOLUTION. After incubation, rinse in PBS (2 min, 2 times) (Incubation time: 30 min) 2. BLOCKING SOLUTION: Reagent 3 (Ready-to-use). • Apply sufficient quantity to cover specimen. Incubate at room temperature. Drain or blot off the solution. DO NOT RINSE. (Incubation time: 10 min) 3. BIOTINYLATED SHEEP ANTI- BrdU: Reagent 4 (Ready-to-use). • Add sufficient antibody to cover specimen. Incubate at room temperature. Rinse with PBS (2 min, 2 times). (Incubation time: 60 min) 4. STREPTAVIDIN-PEROXIDASE: Reagent 5 (Ready-to-use). • Add sufficient antibody to cover specimen. Incubate at room temperature. Rinse with PBS (2 min, 2 times). (Incubation time: 10 min) 5. DAB MIXTURE: Add 1 drop (40 µl) of concentrated DAB substrate to 1 ml of reaction mix 6A. Mix well. Protect from light and use within 1 h. • Apply 2 or more drops of DAB MIXTURE to each section. Incubate. Rinse well with distilled water. If running less than 10 slides it may be helpful to remove the dropper bottle tips and pipet 4 µl of DAB concentrate for every 100 µl of substrate (Incubation time: 2-5 min) 6. HEMATOXYLIN: Reagent 7 (Ready-to use). • Counter stain with sufficient quantity of HEMATOXYLIN to cover specimens. Wash slides in tap water. Put slides into PBS until sections turn blue (~30 s). Rinse in distilled water. (Incubation time: 1-2 min) 7. MOUNTING MEDIA: Reagent 8 (Ready-to-use). • Dehydrate slides in 90% ethanol (10 seconds, 2 times), 100% ethanol (10 seconds, 2 times) and xylene (10 seconds, 2 times). Add 2 drops of MOUNTING media and coverslip.
      Detailed protocolA. Paraffin-Embedded Tissues
      Preparation of Slides:
      1. Tissues should be labeled with BrdU.
      2. Fix target tissue in 10% BF (buffered formalin), or in alcohol-based fixative (such as alcohol or methacarn). Process tissues for paraffin embedding.
      3. Cut tissue sections (3-4 microns) and place on previously subbed slides (e.g., poly-L-lysine coated). Dry slides in a 60°C oven for 30-60 min.
      4. Deparaffinize slides in 2 changes of xylene, 5 min each. Rehydrate slides in a series of graded alcohols (100% for 5 min, then 90%, 80%, and 70% for 3 min each).
      5. Formalin fixed tissues require either trypsin digestion or other pretreatment as described in reference 4 above.
      6. (Optional): If alcohol-fixed tissues are used, tissues sections can be post-dipped in 10% buffered formalin for 30-60 s. Rinse well. This may improve morphology.

      B. Cultured Cells and Cell Suspensions
      Preparation of Slides:
      1. Label cells with 10 µM BrdU for 30 min.
      2. Remove labeling medium from cells and wash in several changes of PBS.
      3. Fix cells in 70% alcohol or acid-ethanol for 15-30 min at 4°C, then air dry. Acetone or methacam fixatives also can be used. Staining will be best if slides are used immediately, but slides can be stored at 4°C for up to a week if needed.
      4. If necessary, block for endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 min.
      5. Wash in 3 changes of distilled water for 2 minutes each.