TACS TM TdT Kit. In Situ Apoptosis Detection Kit. Catalog Number: TA4629. Replenisher Kit. 30 tests

Transcription

1 TACS TM TdT Kit In Situ Apoptosis Detection Kit Catalog Number: TA4629 Replenisher Kit 30 tests This package insert must be read in its entirety before using this product. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

2 Contents TABLE OF CONTENTS Page PRINCIPLE OF THE ASSAY 2 REAGENTS PROVIDED MATERIALS REQUIRED BUT NOT PROVIDED 4 Reagents Equipment 4 Disposables PROCEDURE 5 WARNINGS Hazardous Ingredients 6 Handling Precautions Emergency Exposure Procedures 6 MANUFACTURED FOR AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) McKinley Place N.E. (612) Minneapolis, MN FAX: (612) United States of America info@rndsystems.com R&D Systems Europe 19 Barton Lane TELEPHONE: (0) Abingdon Science Park FAX: (0) Abingdon, Oxon OX14 3NB info@rndsystems.co.uk United Kingdom R&D Systems GmbH FREEPHONE: (0) Borsigstrasse 7 TELEPHONE: (0) Wiesbaden-Nordenstadt FAX: (0) Germany infogmbh@rndsystems.co.uk

3 PRINCIPLE OF THE ASSAY During apoptosis, specific calcium-dependent endonucleases degrade genomic DNA creating fragments with double-stranded breaks. TACS in situ Apoptosis Detection Kits are used to identify apoptotic cells by detecting DNA fragmentation through a combination of enzymology and immunohistochemistry techniques. The TACS TdT Replenisher Kit is a replacement of the limiting reagents within the TACS in situ Apoptosis Detection Kits (DAB TA4625, TACS Blue Label TM TA4626 and Fluorescein TA4627). This kit contains the TdT enzyme and nucleotide mix, proteinase K, cation solutions for optimizing labeling, and a streptavidin-hrp conjugate. Cell and tissue samples are first fixed to prevent the loss of low molecular weight DNA fragments. To make the DNA accessible to the labeling enzyme, the cell membranes are permeabilized with proteinase K (or Cytonin reagent, cat# ). Endogenous peroxidase activity is quenched using hydrogen peroxide. Next, biotinylated nucleotides are incorporated into the 3-OH ends of the DNA fragments by Terminal deoxynucleotidyl Transferase (TdT). The biotinylated nucleotides are then detected by using streptavidin-horseradish peroxidase conjugate followed by a substrate, such as diaminobenzidine (DAB) or TACS Blue Label or by using a streptavidin-fluorescein conjugate. Contact R&D Systems for more information on additional apoptosis detection products for the study of apoptosis and cell death. 2

4 REAGENTS PROVIDED The TACS TdT Replenisher Kit is a replacement of the limiting reagents in the TACS TdT in situ Apoptosis Detection Kits. This kit does not contain all of the reagents included in the complete TACS TdT kits or the appropriate detection protocol. TA4629 TdT in situ Replenisher Product Code Component Quantity Storage Conditions Proteinase K Solution 30 µl -20 C TdT-dNTP Mix 30 µl -20 C TdT Enzyme 30 µl -20 C Streptavidin-HRP 1 30 µl 2-8 C X Co µl -20 C X Mg µl -20 C X Mn µl -20 C 1 Contains 0.05% Thimerosal 3

5 MATERIALS REQUIRED BUT NOT PROVIDED Reagents TdT in situ Apoptosis Detection Kit (DAB TA4625, TACS Blue Label TA4626, Fluorescein TA4627) and appropriate detection protocol (included within the specific kit) 10X phosphate-buffered saline (PBS), ph 7.4 (Ca 2+ and Mg 2+ -free) 37% formaldehyde 95% and 100% ethanol or denatured alcohol Deionized, sterile water 30% hydrogen peroxide o- or p-xylene Mounting medium Equipment 37 C incubator -20 C and 2-8 C storage Coplin staining jars 57 C incubator or slide warmer (for paraffin sections) Refrigerated centrifuge (for cell sample preparation) Adjustable pipettors (0-20 µl, µl, µl) and tips Standard light microscope Cryostat Humidity chamber Ice bucket 50 and 500 ml graduated cylinders Disposables Glass slides, pre-treated for electrostatic adherence 50 ml tubes L, µl pipette tips microcentrifuge tubes 1.5 ml and 10.0 ml serological pipettes Gloves 4

6 PROCEDURE Refer to the detailed procedure for apoptosis detection listed in the TACS TdT in situ Apoptosis Detection Kit product insert for either the DAB (TA4625), TACS Blue Label (TA4626) or Fluorescein (TA4627) kit. These inserts will provide the necessary instructions for preparing reagents, samples and controls, labeling, counterstaining and detection, and a troubleshooting guide. 5

7 WARNINGS Hazardous Ingredients The acute and chronic effects of overexposure to reagents of this kit are unknown. Some kit reagents contain minute amounts of thimerosal, which as a concentrated solution may be fatal if swallowed, inhaled, or absorbed through the skin. Thimerosal contains mercury; abide by local regulations for handling and disposal. Handling Precautions Safe laboratory procedures should be followed when handling all kit reagents. It is recommended that protective laboratory clothing and equipment (gloves, laboratory coat, safety glasses) be worn when handling kit reagents. Emergency Exposure Procedures In case of exposure to reagent solutions, we recommend following these emergency first-aid procedures: Skin or eye contact Wash with water for at least 15 minutes. Remove any contaminated clothing. Inhalation Remove individual to fresh air. If breathing is difficult, give oxygen and call a physician. Ingestion Rinse mouth with copious amounts of water and call a physician. TACS and Cytonin are trademarks of Trevigen, Inc. 6

8 NOTES /01 7