HCS30 BrdU Immunohistochemistry System

HCS30
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Microscopy

      Pricing & Availability

      Catalog Number AvailabilityPackaging Qty/Pack Price Quantity
      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 mouse 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 mouse 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

          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

          Revision06-May-2016 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 Mouse 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 MOUSE 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 MOUSE 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.
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.