TACS TM TdT Kit. In Situ Apoptosis Detection Kit. Catalog Number: TA4625 DAB. 30 tests FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Transcription

1 TACS TM TdT Kit In Situ Apoptosis Detection Kit Catalog Number: TA4625 DAB 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 HINTS TO OPTIMIZE ACCURACY REAGENTS PROVIDED 6 MATERIALS REQUIRED BUT NOT PROVIDED Reagents 6 Equipment PROCEDURES 7 Sample Preparation and Fixation 7 Preparation of Reagents Permeabilization 13 Quenching of Endogenous Peroxidase Activity TdT Labeling 14 Detection and Counterstaining CONTROLS 15 INTERPRETATION OF RESULTS 16 TROUBLESHOOTING GUIDE APPENDICES 18 Reagent and Buffer Composition Fixation Methods 19 Double Labeling Hints and Tips Assembling a Humidity Chamber 21 TACS Nuclease Treated Control Analysis by Electron Microscopy 22 Labeling Suggestions WARNINGS 23 Hazardous Ingredients 23 Handling Precautions Emergency Exposure Procedures 23 Reactivity Data MANUFACTURED FOR AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) McKinley Place NE (612) Minneapolis, MN FAX: (612) United States of America info@rndsystems.com R&D Systems Europe, Ltd. 19 Barton Lane TELEPHONE: +44 (0) Abingdon Science Park FAX: +44 (0) Abingdon, OX14 3NB info@rndsystems.co.uk United Kingdom R&D Systems GmbH Borsigstrasse 7 TELEPHONE: +49 (0) Wiesbaden-Nordenstadt FAX: +49 (0) Germany infogmbh@rndsystems.co.uk R&D Systems Europe 77 boulevard Vauban FREEPHONE: LILLE CEDEX FAX: France info@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. All reagents necessary for labeling DNA fragments within apoptotic cell or tissue samples are included in the kit. 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 TM reagent. Cytonin is optimized to permeabilize cells and proteinase K is optimized for the permeabilization of tissues prior to in situ detection of apoptosis. 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 detected by using streptavidin-horseradish peroxidase conjugate followed by the substrate, diaminobenzidine (DAB). The enzyme reaction generates an insoluble colored precipitate where DNA fragmentation has occurred. To discriminate apoptotic cells from necrotic cells, the samples are counterstained to aid in the morphological verification of apoptosis. DAB-stained samples are examined using a light microscope. 2

4 HINTS TO OPTIMIZE ACCURACY Read this section before proceeding. 1. Components that require -20 C storage, must be placed in a manual defrost freezer. 2. Select an appropriate fixation method. The choice of fixative will affect the overall staining results. It is essential to select a ph neutral fixative that will maintain tissue morphology and DNA integrity. We recommend the use of 3.7% formaldehyde solution. Other fixatives, such as 4% paraformaldehyde without methanol, 4% formaldehyde, Bouin s fixative and glutaraldehyde may be used. Fixatives containing acid or bases (e.g., trichloroacetic acid) should be avoided since they cause DNA damage that translates into high background. Note: Bouin s fixative may interfere with methyl green counterstaining. 3. Minimize the length of fixation. The amount of time required for fixation varies with the type of specimen. In general, fixation times between 10 minutes (for cells and cryosections) and 8 hours (for larger or less porous tissues) are sufficient. At least 10 volumes of fixative relative to the tissue size should be used. For longer fixation times, the fixative should be changed at least once after the first hour. Tissues that have been stored long-term in fixative may not label because the 3 -OH ends of the DNA may be oxidized. 4. Adjust incubation times. Use the suggested times for permeabilization treatment (proteinase K or Cytonin) and enzyme labeling (TdT) for your first experiment. If necessary, optimize by increasing or decreasing the reagent incubation time depending on your specific samples. 5. Proteinase K is temperature sensitive. Room temperature can affect the efficiency of proteinase K treatment. Maintain a temperature range of C to minimize assay variability. 6. Keep samples covered with reagent. Use coverslips to ensure proper reagent coverage of samples. Keep slides level. Do not reuse coverslips. The coverslip may be removed by dipping the slide into a clean beaker of the appropriate buffer or solution. This causes the coverslip to float free. 7. Do not allow samples to dry during the staining procedure. Use a humidity chamber during the 37 C incubation step (refer to the appendix, page 21). 8. Maintain TdT enzyme at -20 C. The TdT enzyme should not be held on ice. For best results, remove the vial of TdT from the -20 C freezer, pipette the required amount and immediately return to the -20 C freezer. Alternatively, the enzyme can be placed into a -20 C storage block specifically designed for use at the laboratory bench. TdT is supplied in glycerol, which is liquid at -20 C but not at -80 C. Therefore, storage of the TdT enzyme in a -80 C freezer during routine use is not recommended. TdT enzyme may be placed in a -80 C freezer for long term storage, but cycling between a -20 C and a -80 C freezer should be avoided. 3

5 9. Use high quality water. DNase-free or molecular biology grade water should be used until the specimens have been labeled. Deionized water is recommended. It may be useful to test the water with the control slides prior to labeling the samples. 10. Use treated slides. To maintain tissue and cell adhesion to the slide during processing, use glass slides pre-treated for electrostatic adherence. 11. Be certain that you can identify which side of the slide your specimen is on. Label the slides appropriately. When using slides to grow adherent cells, label the slides prior to autoclaving. 12. Use Coplin staining jars. All washes and staining procedures should be performed in 50 ml Coplin staining jars, unless otherwise indicated. A 50 ml polypropylene conical tube may be substituted, however, a maximum of 2 slides can be processed per tube, and the specimens MUST face away from each other. Do not store xylenes in polypropylene tubes. 13. Optimize TdT labeling using the cations included in the kit. The TdT kits include a choice of 3 divalent cations (Co 2+, Mn 2+, Mg 2+ ) that may be used to modulate the TdT enzymatic reaction. Generally, staining may be enhanced by using Mn 2+ and background staining may be reduced by using Mg 2+. Begin optimization using Co Prepare the TdT reaction mix in the correct sequence. Add the nucleotide mix, the divalent cation and the enzyme prior to adding the 1X TdT Labeling Buffer. 15. Use fresh hydrogen peroxide. Hydrogen peroxide (H 2 O 2 ) loses activity during storage. To ensure activity, purchase H 2 O 2 in small volumes and store no longer than 2 months. Do not store or reuse diluted H 2 O Quench endogenous peroxidase to eliminate background staining. Some tissues and cells contain high levels of endogenous peroxidase that may not be sufficiently quenched using 2% H 2 O 2. In this case, quenching for 5 minutes using 3% H 2 O 2 in methanol may be substituted. Longer quenching times may be tried; however, the DNA in tissue sections and cells may be oxidized by prolonged H 2 O 2 treatment. 17. Hydrogen peroxide causes DNA strand breaks. After H 2 O 2 quenching, immediately transfer slide(s) into 1X PBS. 18. Optimize the counterstaining procedure for specimens prior to the first use of the kit. Use an extra cell sample or tissue section to determine the optimal time for counterstaining before you label any samples. 4

6 19. Thaw the DAB solution. The DAB solution must be thawed prior to pipetting. Place in a 37 C waterbath for at least 10 minutes prior to use. 20. DAB is a suspected carcinogen. Use appropriate handling and disposal procedures. 21. For best results, immobilize suspension cells on slides immediately after collection. The use of fresh cells is recommended. However, some cells may be stored in 80% ethanol for up to 2 months at 2-8 C. This must be empirically determined for each cell type. 22. Deposition of cells by cytospin is not recommended. Preparation of non-adherent (suspension) cells by cytospin may rupture cells and interfere with the visualization of apoptotic morphologies. 23. For best results, use 5 µm sections. Paraffin-embedded sections may be 3-6 µm; cryosections may be 5-10 µm. 24. To retain optimal integrity of samples, do not section until shortly before use. 25. When working with specimens from formaldehyde-perfused animals, use water (or other low-ionic strength solution), not PBS, to float sections for collection to ensure those sections adhere to the slides. 5

7 REAGENTS PROVIDED TA4625 TdT - DAB Kit Component # Component Quantity Storage Conditions Proteinase K Solution 30 µl -20 C X TdT Labeling Buffer ml 2-8 C X TdT Stop Buffer 100 ml 2-8 C TdT dntp Mix 30 µl -20 C TdT Enzyme 30 µl -20 C Streptavidin-HRP 2 30 µl 2-8 C DAB Solution ml -20 C Methyl Green Stain (1.0%) 50 ml room temperature X Co µl -20 C X Mg µl -20 C X Mn µl -20 C Cytonin 5 ml 2-8 C TACS-Nuclease 15 L 2-8 C TACS-Nuclease Buffer 1.5 ml 2-8 C 1 Contains 0.01% Thimerosal 2 Contains 0.05% Thimerosal 3 Contains Diaminobenzidine (DAB), a suspected carcinogen Prior to opening the vial, centrifuge for seconds at 3000 rpm. MATERIALS REQUIRED BUT NOT PROVIDED Reagents 10X and 1X phosphate-buffered saline (PBS), ph 7.4 (Ca 2+ and Mg 2+ -free) 37% formaldehyde 100% ethanol (reagent grade) 95% ethanol (reagent grade) Mounting medium Methanol 30% hydrogen peroxide (H 2 O 2 ) Xylenes (histological grade) 1-butanol (for Methyl Green Counterstain, protocol B) Deionized water, DNase-free 6

8 Equipment Glass slides, pre-treated for electrostatic adherence Light microscope 37 C incubator Coplin staining jars (50 ml conical tubes may be substituted) 57 C incubator or slide warmer (for paraffin sections) Refrigerated centrifuge (for cell sample preparation) Adjustable pipettors (0-20 µl, µl, µl) and tips PROCEDURES Sample Preparation and Fixation Suspension Cells 1. Prepare 3.7% formaldehyde solution: 37% Formaldehyde 5 ml 10X PBS DNase-free water 5 ml 40 ml 2. Collect the cells by centrifugation at 500 x g for 10 minutes at 2-8 C. For some cell types, centrifugation speed may need to be adjusted. Gently resuspend the cell pellet in 3.7% formaldehyde solution at 1 x 10 6 cells/ml. Incubate for 10 minutes at room temperature. 3. Collect the cells by centrifugation at 500 x g for 10 minutes at room temperature. Gently resuspend the cells in 80% ethanol at a concentration of 1 x 10 6 cells/ml. The use of fresh cells is recommended, but some cells may be stored for up to 2 months at 2-8 C. This must be empirically determined for each cell type. Do not freeze. 4. Pipette 100 µl of the cells onto a clean glass slide, pre-treated for electrostatic adherence. Dry at room temperature for 5-10 minutes or until no moisture is evident. Label the slide so that the side of the slide that contains the specimen can be easily identified. Glass slides pretreated for electrostatic adherence are recommended. Other pretreatments (e.g. gelatin) can cause increased background staining. 5. Place the slides with completely dried cells into 50 ml of 70% ethanol for 5 minutes at room temperature. Remove and air dry for 1-2 hours at C. The slide is now ready for staining. Dried, unstained slides may be stored at 2-8 C with desiccant for up to six months. 6. For Labeling, proceed to Permeabilization, (page 13). For Preparation of Reagents, see page 10. 7

9 Adherent Cells 1. Transfer a sterile, labeled slide into a tissue culture dish. Seed with cells. Cells should be grown and treated as per the normal protocol. 2. Prepare 3.7% formaldehyde solution: 37% Formaldehyde 5 ml 10X PBS DNase-free water 5 ml 40 ml Tissues 3. Remove the slide from the dish without disturbing the cell monolayer. 4. Wash the slide in 50 ml of 1X PBS. 5. Place the slide in 50 ml of 3.7% formaldehyde solution. Incubate for 10 minutes at room temperature. 6. Wash the slide in 50 ml of 1X PBS. 7. For Preparation of Reagents, see page 10. If cells fall off the slide during the labeling procedure, or if storage is desired, transfer the slide into 50 ml of 70% ethanol. Incubate for 5 minutes at room temperature. Remove the slide and air dry for 1-2 hours at C. Unstained slides may be stored with desiccant at 2-8 C for up to 6 months. For labeling, proceed to Permeabilization, page 13. The following information should be used as a guideline. The method of fixation depends upon the tissue type and origin. For best results, prepare 5 µm sections. Paraffin sections may be 3-6 µm; cryosections may be 5-10 µm. Paraffin-Embedded Tissues Fixation and Paraffin Embedding 1. Prepare 3.7% formaldehyde solution: 37% Formaldehyde 5 ml 10X PBS DNase-free water 5 ml 40 ml 2. Immerse the tissues in 3.7% formaldehyde solution immediately after dissection. Fix for 1-8 hours. After the first 30 minutes of incubation, transfer to fresh 3.7% formaldehyde solution and incubate 30 minutes to 7.5 hours more. 3. Transfer the tissues to 75% ethanol and wash for 1 hour at room temperature. Repeat the wash using fresh 75% ethanol. 8

10 4. Transfer the tissues to 95% ethanol and wash for 1 hour at room temperature. Repeat the wash using fresh 95% ethanol. 5. Transfer the tissues to 100% ethanol for 1 hour at room temperature. Repeat the wash using fresh 100% ethanol. 6. Transfer the tissues to xylenes and wash for 1 hour at room temperature. Repeat the wash using fresh xylenes. 7. Embed the tissues in molten paraffin wax (58 C) for 1 hour. Embed again, using new paraffin wax. Paraffin-embedded specimens can be stored at 2-8 C for up to one year. To retain sample integrity, do not section until shortly before use. 8. Slice the tissue sections using a microtome. Float sections on a 45 C water bath. Mount the sections on clean glass microscope slides, pre-treated for electrostatic adherence. 9. Deparaffinize the tissue sections using the following procedure. Do not deparaffinize until you are ready to proceed to the Labeling Protocol. Deparaffinization of Tissues 1. Heat the slides at 57 C for 5 minutes on a slide warmer to melt wax. 2. Transfer the slides to 50 ml of 100% xylenes. Incubate for 10 minutes at room temperature. 3. Transfer the slides to 50 ml of fresh 100% xylenes. Incubate for 5 minutes at room temperature. 4. Wash slides sequentially for 5 minutes per wash at room temperature in: 50 ml of 100% ethanol; 50 ml of 95% ethanol; and 50 ml of 70% ethanol. 5. Wash the slides twice for 2 minutes per wash in 50 ml of DNase-free water. Use fresh water for each wash. 6. Transfer the slides to 50 ml of 1X PBS and incubate for 10 minutes at room temperature. 7. For Labeling, proceed to Permeabilization (page 13). For Preparation of Reagents, see page 10. Note: The xylenes and ethanol used for deparaffinization of tissues may be reused several times. However, these xylenes and ethanol CANNOT be used for later steps in this staining protocol. 9

11 Cryosections 1. Prepare 3.7% formaldehyde solution: 37% Formaldehyde 5 ml 10X PBS DNase-free water 5 ml 40 ml 2. Immediately following dissection, freeze the tissue samples rapidly using liquid nitrogen or dry ice. 3. Prepare the cryosections at -18 C. Transfer the sections to clean glass microscope slides, pre-treated for electrostatic adherence. Dry for 2 hours at C. 4. Fix in 50 ml of 3.7% formaldehyde solution for 10 minutes at room temperature. 5. Wash the slides 2 times for 5 minutes per wash in 50 ml of 1X PBS. Use fresh 1X PBS for each wash. 6. For Labeling, proceed to Permeabilization, (page 13). For Preparation of Reagents, see below. Preparation of Reagents Reagents marked with an asterisk (*) should be prepared immediately before use. The volumes given for each reagent are based on processing samples of up to 4 cm 2 immobilized on glass slides. Different configurations of chamber slides, culture plates, free-floating sections and the use of glass cover slips may require adjustments to the stated volumes. 1. 1X PBS Refer to the appendix for preparation of 10X PBS. Approximately 500 ml of 1X PBS is used to process 1-10 slides. Dilute 10X PBS to 1X using dh 2 O. Store 1X PBS at room temperature % Buffered Formaldehyde* To prepare, add: 37% Formaldehyde 5 ml 1X PBS 45 ml Wear gloves and exercise caution when handling formaldehyde solutions. Refer to the appendix for alternative fixation methods. 10

12 3. Proteinase K Solution* 50 µl of Proteinase K Solution is used per sample. Thaw provided Proteinase K at room temperature, then place on ice until use. Immediately before use, add: dh 2 O 50 L Proteinase K 1 L 4. Cytonin If required, 50 µl of Cytonin is used per sample. Cytonin is ready for use. Store at 2-8 C. Discard if solution is cloudy. 5. Quenching Solution* Prepare immediately before use. 50 ml of Quenching Solution is used to process 1-10 slides. To prepare, add: Methanol 45 ml 30% H 2 O 2 5 ml Always use fresh 30% H 2 O 2. It is recommended that 6 ml aliquots of fresh 30% H 2 O 2 be made and stored at 2-8 C. For each labeling procedure, use a fresh 30% H 2 O 2 aliquot and discard the unused portion. 6. 1X TdT Labeling Buffer 50 ml of 1X Labeling Buffer is used to process 1-10 slides. Dilute the 10X TdT Labeling Buffer to 1X using dh 2 O. Store at room temperature until use. Remove an aliquot of 50 µl per sample for preparing the Labeling Reaction Mix and place on ice. 7. Labeling Reaction Mix* Thaw TdT-dNTP Mix at room temperature, then place on ice. To maintain optimal enzyme activity, remove the TdT Enzyme from the freezer only long enough to pipette the required volume. Alternatively, place the TdT Enzyme in a -20 C freezer block. Prepare the Labeling Reaction Mix just before use and keep the prepared reaction mix on ice. Prepare 50 µl per sample: TdT-dNTP 1 L 50X Cation Stock 1 L TdT Enzyme 1 L 1X TdT Labeling Buffer (step 6) 50 L Note:To select the appropriate Cation stock, see the appendix (page 22). 8. 1X TdT Stop Buffer 50 ml of 1X TdT Stop Buffer is used to process 1-10 slides. Dilute the 10X TdT Stop Buffer to 1X using dh 2 O. Store at room temperature until use. 11

13 9. Streptavidin-HRP Solution* 50 µl of Streptavidin-HRP Solution is used per sample. Store prepared solution at room temperature until use. To prepare, dilute: 1X PBS 50 L Streptavidin-HRP 1 L 10. DAB Solution* 50 ml of DAB Solution is used to process 1-10 slides. Thaw DAB at 37 C, then place at room temperature. Note: Do not place on ice, or the DAB solution will precipitate. Prepare DAB Solution no more than 20 minutes before use. To prepare, add: 1X PBS 50 ml DAB 250 L 30% H 2 O 2 50 L Use only fresh 30% H 2 O 2. It is recommended that 6 ml aliquots of fresh 30% H 2 O 2 are made and stored at 2-8 C. For each labeling procedure, use a fresh aliquot, then discard any remaining solution. 11. Methyl Green Methyl Green is ready for use. Methyl Green can be reused many times. Store in a closed container to prevent evaporation. If a precipitate forms, filter sample through Whatman 3MM paper. 12. Xylenes Mixed xylenes can be used for deparaffinization and for clarification prior to mounting cover slips onto the samples. Xylenes used for deparaffinization may be reused several times. Xylenes used in deparaffinization should not be used for clarification %, 95%, 70% ethanol Either 100% (200 proof) or denatured alcohol (90% ethanol, 5% methanol, 5% isopropanol) may be used. Dilute with deionized H 2 O to prepare 95% and 70% solutions. Ethanol used for deparaffinization may be reused several times. Ethanol used in deparaffinizations should not be used for dehydration. 14. TACS-Nuclease and Buffer* TACS-Nuclease should be diluted 1:50 in TACS-Nuclease Buffer just prior to use and kept on ice. For each control, prepare: TACS-Nuclease Buffer 50 L TACS-Nuclease 1 L 12

14 Permeabilization Rehydrate Samples (if applicable) (a) Wash the slides sequentially for 5 minutes per wash at room temperature in: 50 ml of 100% ethanol; 50 ml of 95% ethanol; and 50 ml of 70% ethanol. (b) Place the slides in 50 ml of 1X PBS. Incubate for 10 minutes at room temperature. (c) Carefully dry the slides around each sample with a lab wipe. Do not allow the samples to dry. Permeabilize Samples Use the Proteinase K protocol for most tissue samples and for control slides. Use the Cytonin protocol for freshly prepared cell samples. Proteinase K digestion: (a) Place 50 µl of the Proteinase K solution onto each sample. Cover with a coverslip. (b) (c) Incubate at room temperature for 5 minutes (cells) or 15 minutes (tissues). Wash the slides twice for 2 minutes per wash in 50 ml of DNase-free water. Use fresh water for the second wash. If preparing a positive control, treat the sample with TACS-Nuclease (see page 21) prior to proceeding to step d. (d) Proceed immediately to Quenching of Endogenous Peroxidase (page 14). Incubate for at least one minute. Note: Overtreatment with Proteinase K may cause extensive damage and/or loss of cells and tissues. However, if your sample does not generate a positive result, you may need to increase the length of the Proteinase K treatment (up to 15 minutes for cells or up to 1 hour for tissues) or increase the incubation temperature (up to 37 C). Cytonin treatment: (a) Place 100 µl of Cytonin onto each sample. Cover with a coverslip. (b) (c) Incubate at room temperature for minutes. Wash the slides twice for 2 minutes per wash in 50 ml DNase-free water. Use fresh water for the second wash. If preparing a positive control, treat the sample with TACS-Nuclease (see page 21) prior to proceeding to step d. (d) Proceed to Quenching of Endogenous Peroxidase (page 14). 13

15 Quenching of Endogenous Peroxidase 1. Place the slides into 50 ml of Quenching Solution. Incubate at room temperature for 5 minutes. Note: Do not leave longer than 5 minutes. Longer quenching times may be tried; however, DNA from cells and tissue sections may be damaged. 2. Remove the slides from the Quenching Solution. Shake or tap to remove excess liquid. Transfer the slides into 50 ml 1X PBS for 1 minute. Immerse slides in the 1X TdT Labeling Buffer and incubate for 5 minutes. Proceed to TdT Labeling. TdT Labeling 1. Carefully dry the glass slide around each sample. Do not allow the samples to dry. 2. Pipette 50 µl of Labeling Reaction Mix onto each sample. Cover with a coverslip. 3. Place the slides in a humidity chamber in a 37 C incubator. Incubate for 1 hour. Refer to the appendix (page 21) to construct a humidity chamber. 4. Transfer the slides into 50 ml of 1X TdT Stop Buffer. Incubate for 5 minutes at room temperature. 5. Wash slides 2 times in 1X PBS for 2 minutes each. Proceed to the Detection Step below. Diaminobenzidine (DAB) Detection and Counterstaining Note: DAB is a suspected carcinogen. Use appropriate handling and disposal procedures. 1. Carefully dry the slide around each sample with a lab wipe. Do not allow the sample to dry. 2. Pipette 50 µl of the Streptavidin-HRP Detection Solution onto each sample. Cover with a coverslip. Incubate at room temperature for 10 minutes. 3. Wash the slides 2 times (2 minutes per wash) in 50 ml of 1X PBS at room temperature. Use fresh 1X PBS for the second wash. The slides must remain in the second wash if the DAB solution is not ready. 4. Transfer the slides to 50 ml of DAB Working Solution. Incubate at room temperature for 2-10 minutes. The reaction can be monitored by dipping the slide in water and viewing it under a microscope. Do not exceed 10 minutes in the DAB Working Solution; background staining may occur. 5. Rinse the slides in 50 ml dh 2 O. Transfer to fresh water and wash for a few seconds. Note: Dispose of DAB according to local regulations. 6. Methyl Green Counterstain Staining varies with cell or tissue type and must be determined empirically. Begin with protocol A, which is useful for most cells and tissues. If counterstaining is unsatisfactory, protocol B may provide improved results. Optimize the counterstaining procedure for specific specimens on an extra sample prior to using the TdT in situ Apoptosis Detection Kit. Use protocol B for tissue specimens prepared with Bouin s fixative. 14

16 Methyl Green Counterstain Protocol A: 1. Transfer the slides to 50 ml of Methyl Green solution. Incubate at room temperature for 5 seconds - 5 minutes. 2. Wash the slides sequentially by dipping 10 times each in: dh 2 O; 70% ethanol, 2 changes; 95% ethanol, 2 changes; 100% ethanol, 2 changes; and Xylenes, 2 changes. 3. Wipe off the excess xylenes from the back surface of each slide. Leave the xylenes on the top of the slides; it aids in the mounting process. 4. Place 1 drop (approximately L) of mounting media on each sample. 5. Starting on one edge, place a glass coverslip onto each slide. Apply gentle even pressure to expell air bubbles. 6. Leave slide flat overnight to allow mounting media to harden. Store slides in the dark. Methyl Green Counterstain Protocol B: 1. Transfer the slides to 50 ml of Methyl Green solution. Incubate at room temperature for 5 minutes. 2. Wash the slides sequentially by dipping 10 times each in: 1-Butanol, until sample turns from blue to green; 1-Butanol, 1 time to rinse; and Xylenes, 2 times (5-10 seconds each) 3. Wipe off the excess xylenes from the back surface of each slide. Leave the xylenes on the top of the slides; it aids in the mounting process. 4. Place 1 drop (approximately L) of mounting media on each sample. 5. Starting on one edge, place a coverslip onto the slide. Apply gentle even pressure on the coverslip, forcing air bubbles out. 6. Leave slide flat overnight to allow mounting media to harden. Store slides in the dark. CONTROLS Prepared control slides are available from R&D Systems (cell culture control slides, Catalog # or tissue control slides, Catalog # ). Experimental controls that should be included when performing the protocol for the first time are listed below: TACS-Nuclease-treated Control After incubation in Cytonin or Proteinase K Solution and washing, treat 1 sample with TACS-Nuclease to generate DNA breaks in every cell (see appendix, page 21). Incubation times may range from 5-60 minutes depending on the cellularity of the tissue. A 15 minute incubation time is recommended as a good starting point. Washing samples with PBS will stop the reaction. The TACS-Nuclease-treated Control will confirm that the permeabilization and labeling reaction has worked. The information can help optimize the conditions for the labeling procedure. The majority of cells should exhibit pale brown nuclear staining. 15

17 Unlabeled Experimental Control The TdT Enzyme should be omitted from the Labeling Reaction Mix for one sample. This control will indicate the level of background labeling (DAB) associated with non-specific binding of the Streptavidin-HRP. This control should not have any brown staining. Experimental Negative Control An appropriate experimental control should be included in each experiment and will depend upon the system under study. Typically, the Experimental Negative Control will be untreated sample, or normal cells/tissues. Many normal or untreated cells and tissues will have a small number of apoptotic cells so a few cells will exhibit positive (brown) staining. Counterstaining Controls Although uncommon, some cells and tissues may take up excessive amounts of the Methyl Green counterstain, obscuring the brown DAB staining. It is recommended that one or two samples are processed up to and including the dh 2 O wash step after the Quenching Step of the Labeling Procedure. Process through counterstaining. Staining times of 5 seconds to 5 minutes have been noted. Start with a 30 second immersion in Methyl Green counterstain and alter accordingly. INTERPRETATION OF RESULTS Apoptosis is defined by morphological criteria. The morphological data obtained from standard microscopy and histochemistry should always be considered in conjunction with biochemical assays to confirm apoptosis. Methyl Green allows all cells in the specimen to be visualized. Cells that are condensed (pyknotic, mitotic or apoptotic) will exhibit increased Methyl Green uptake. Cells containing fragmented nuclear chromatin characteristic of apoptosis will exhibit a brown nuclear staining that may be very dark after labeling. This dark brown staining is typically associated with cell condensation. Brown staining in the cytoplasm as well as the nucleus of enlarged or swollen cells may occur in instances of necrosis. In tissue sections where cells have been torn open during sectioning or the edges of the specimen are ragged, there may be non-specific brown staining that is not associated with nuclei. The suggested experimental controls are important for data interpretation. The controls allow optimization of in situ detection of apoptosis without expending valuable test samples. Under optimal conditions, the Unlabeled Control should show no brown staining, the TACS-Nuclease-treated Control should show pale brown staining in almost all cells, and the Experimental Negative Control should have less than 20% brown-stained cells. The brown staining of TACS-Nuclease-treated cells is paler and usually more diffuse than the staining of truly apoptotic cells. This is due to the difference in chromatin structure between Nuclease-treated normal cells and the fragmented chromatin of apoptotic cells. The Counterstain Control should show a pale green staining of all cells with some variability in intensity between cell types and darker staining of any condensed cells within that sample. Refer to the Troubleshooting Guide for information if the controls do not provide the expected result. 16

18 TROUBLESHOOTING GUIDE Problem Possible Cause Solution No staining of cells Cells are not apoptotic. Conditions for inducing apoptosis may need to be changed. These conditions vary with cells. Verify that cells are undergoing apoptosis by DNA laddering or by identification of the morphological features of apoptosis (e.g., chromatin condensation, presence of apoptotic bodies, cellular condensation). Cells are impermeable to reagents. DNA is hydrolyzed due to poor storage of samples. Reagents are no longer functional due to improper storage or the age of the reagents. Test to make sure that sample is sufficiently permeabilized by labeling a positive control generated by TACS-Nuclease treatment (see page 21). Use fresh samples. If using fixed sections, do not cut until shortly before use. Confirm that reagents were stored at the correct storage temperatures. Faint or light staining of cells Dark cytoplasmic staining Sample requires a longer enzyme labeling step. Sample requires a longer streptavidin incubation time. Not enough streptavidin-hrp bound to sample. Too much streptavidin-hrp bound to sample. Extend the TdT labeling reaction up to 2 hours. Use a humidity chamber to prevent drying of samples. Increase the streptavidin-hrp binding step up to 30 minutes at room temperature. Use a humidity chamber to prevent drying of sample. Increase the concentration of streptavidin-hrp. Dilute the streptavidin-hrp reagent from 2-10 fold more, or reduce the streptavidin-hrp incubation time. 17

19 Problem Possible Cause Solution Dark cytoplasmic staining (continued) Cells slough off slide during treatment Non-specific binding of streptavidin-hrp to sample. Detection step requires a shorter incubation time. Endogenous peroxidase activity is not sufficiently quenched. Cells are in a late stage of apoptosis or necrosis. Sample dried out during labeling procedure. Excessive proteinase K treatment Insufficient drying of samples prior to staining Increase the number of washes after streptavidin-hrp incubation step. Prepare streptavidin-hrp in 1X PBS + 1% bovine serum albumin. Decrease the substrate incubation time. Use freshly prepared H 2 O 2 solution. Substitute 3% H 2 O 2 in methanol. Perform a time-course and/or dose-response experiment. Use coverslips and carry out incubation steps in a humidity chamber. Decrease the proteinase K incubation time or dilute the proteinase K solution. Use Cytonin instead of proteinase K. Treat the samples in 70% ethanol, and dry for 1-2 hours prior to the reaction. APPENDICES Reagent and Buffer Composition 10X PBS, ph 7.4 (not provided in the kit) 75 mm sodium dihydrogen phosphate 25 mm disodium hydrogen phosphate 1.45 M sodium chloride 10X TdT Labeling Buffer 1 M TACS Safe-TdT Buffer 0.5 mg/ml BSA (RIA grade) 0.6 mm 2-mercaptoethanesulfonic acid (MESNA) 10X TdT Stop Buffer 0.1 M EDTA, ph 8.0 TdT dntp Mix 0.25 mm biotinylated dntp 18

20 Cytonin Permeabilization and blocking agent 1.0% Methyl Green Solution 0.1 M sodium acetate, ph % Methyl Green Proteinase K 1 mg/ml Proteinase K TACS-Nuclease Endonuclease TACS-Nuclease Buffer 50 mm Tris-HCl, ph mm magnesium chloride 100 µg/ml BSA Fixation Methods Formaldehyde is the recommended fixative based on laboratory testing. Other fixatives that maintain DNA integrity may also be used. These include other cross-linking agents such as paraformaldehyde and glutaraldehyde. Regardless of the fixative used, it is important not to fix cells and tissues for extended periods of time. Fixation method will likely be dictated by immunocytochemistry protocols in double labeling experiments (see below). Post-fixation in acetone, ethanol or methanol is common in preparation of tissues and is usually compatible with TACS TdT - DAB. To store the immobilized fixed cells (on slides, chamber slides or cover slips), post-fix in 100% methanol after fixation. Wash in 1X PBS, then store in Cytonin at 2-8 C for up to 1 week. After storage, wash in 1X PBS then continue with the labeling reaction starting with the Quenching Step. Note: If cells are fixed using alcohol (e.g. ethanol), there will be a leakage of small DNA fragments from apoptotic cells during storage and labeling intensity of apoptotic cells will be reduced. Double Labeling Hints and Tips The in situ protocol described here is useful for double labeling experiments when the occurrence of apoptosis can be correlated with expression of cellular antigens. Note: The antibody used must recognize the fixed form of the antigen of interest. The key to double labeling experiments is determining fixation and permeabilization conditions under which both antigen and DNA integrity is maintained. Appropriate fixatives for DNA labeling are listed in the appendix (see above). Post-treatments used in immunocytochemistry to permeabilize or expose antigenic determinants include treatment with proteases, acid or base, detergent and microwaving. Protease treatment is not recommended on most samples because the sample will often disintegrate later during immunocytochemistry or DNA labeling. Strong acid or base treatment should be avoided. Microwaving is an option that has given excellent results in double labeling experiments, but requires careful empirical determination for correct wattage, time and cooling cycles for each sample. 19

21 Empirically determine optimal conditions for immunohistochemistry and in situ detection of apoptosis in separate experiments first. Combine the 2 methodologies only after optimizing separately on the same samples. Plan carefully and include controls to allow interpretation of double-labeled samples. Controls for immunohistochemistry may include omission of primary antibodies to determine binding of the secondary antibody. In addition, blocking the primary antibody binding site with antigens may establish and demonstrate specificity. The selection of the color reaction products should be considered ahead of time. R&D Systems offers Red Label (Catalog # RL) for use with secondary antibodies conjugated to phosphatases. A standard immunohistochemistry protocol is provided below for using phosphatase-conjugated secondary antibody and color development with Red Label Solution. Antibody concentrations, incubation times and temperatures, and buffers may have to be adjusted appropriately for each system under study. Note: Phosphatase-conjugated reagents are inhibited by PBS or other phosphate-containing buffers. Tris buffers should be substituted for PBS. Method: 1. After fixation, permeabilize with Cytonin at room temperature for 1 hour. Use coverslips and a humidity chamber to prevent evaporation. 2. Wash 1 time in 100 mm Tris, ph Incubate at 2-8 C overnight with primary antibody diluted in Cytonin. Use coverslips and a humidity chamber to prevent evaporation. 4. Wash 3 times in 100 mm Tris, ph Incubate with phosphatase-conjugated secondary antibody diluted in Cytonin at room temperature for 1 hour. 6. Wash 3 times in 100 mm Tris, ph Prepare Red Label Solution: a. In a fresh microtube, add 50 µl of dh 2 O and 5 µl of Red Label Solution 1. b. To a new microtube, add 1 µl each of Red Label Solution 2 and Red Label Solution 3, tap microtube to mix. Centrifuge briefly and let stand for 3 minutes at room temperature. Transfer this solution to the microtube from step 7a. 8. Cover sample with prepared Red Label Solution and incubate for up to 30 minutes in the dark. 9. Wash in dh 2 O 3 times to stop the reaction. 10. Proceed with in situ detection (Labeling Procedure) at the Quenching Solution step. 20

22 Many options are available for double labeling experiments. If the antigen is nuclear, carefully select the detection label and counterstains. Labeling nuclear antigens means the signal from the DNA labeling and immunocytochemistry will be in the same subcellular compartment and one signal may obscure the other. Similarly, many counterstains are not compatible with some color reaction products used such as Methyl Green cannot be used with Red Label. Some options are listed below: If a peroxidase-linked secondary antibody is preferred, use Quenching Solution prior to incubation with primary antibody and again prior to in situ detection of apoptosis. DAB Solution may be used for color reaction if alternative peroxidase-based color development is used for detection of apoptosis. The streptavidin-hrp may be replaced with a streptavidin-phosphatase conjugate and developed using a phosphatase-based system such as Red Label. Similarly, fluorescent streptavidin conjugates and secondary antibodies may be used for a fluorescent read-out. Assembling a Humidity Chamber To prevent evaporation, it is recommended that incubations at 37 C are carried out in a humidity chamber. A humidity chamber can be made using a plastic box with a tight-fitting lid and 2 glass rods or other support. Place a paper towel on the bottom of the box and wet thoroughly with water. Lay the glass rods parallel to each other and less than 1 slide length apart on the wet towel. Position the slides on the glass rods and place the plastic box, with lid, in a 37 C incubator. Ensure that the slides are horizontal. TACS-Nuclease Treated Control Treat 1 sample with TACS-Nuclease to generate DNA breaks in the majority of cells. Insert the Nuclease treatment step after permeabilization of the cells. 1. Wash sample in dh 2 O a. 2. Prepare Nuclease solution (50 µl/sample): TACS-Nuclease Buffer 50 µl TACS-Nuclease 1 µl 3. Cover sample with the solution and incubate at 37 C for 5-30 minutes b. 4. Stop the reaction by immersing the sample in 1X PBS. To stop the reaction after treatment, immerse slides in 1X PBS buffer. Nuclease-treated control will confirm that the permeabilization and labeling reaction have worked. The information obtained from the controls can help optimize the conditions for the labeling procedure. The majority of cells should exhibit brown nuclear staining when stained with DAB. a It is important to wash the samples in dh2o before using TACS-Nuclease. b The length of incubation with the Nuclease varies depending upon the type of tissue (e.g. shorter in tissues with low cellularity, such as brain and longer in tissues with high cellularity, such as muscle). 21

23 Analysis by Electron Microscopy The protocol given here can be adapted for electron microscopy. Both pre- and post-embedding labeling can be performed depending on the system under study. For pre-embedding prior to labeling, fix the sample, but post-fix in osmium. After embedding and ultrathin sectioning, process sample for DNA labeling up to and including the washes of the Labeling Procedure prior to incubation with streptavidin. For detection of incorporated biotin, use streptavidin conjugated to colloidal gold and incubate overnight at 2-8 C. Stain with uranyl acetate. For some samples, post-embedding may be more convenient. Use fixed floating sections and process for in situ labeling up to and including the washes in the Labeling Procedure prior to streptavidin binding. Incubate in streptavidin conjugated to colloidal gold overnight at 2-8 C. Wash, then proceed with standard embedding procedure and ultrathin sectioning. Labeling Suggestions for TA4625 The following table shows examples of conditions used for labeling the tissues listed and acts as a guide only. Actual incubation times, permeabilization method, and cation selection may require empirical determination. Brain Heart Lung Liver Kidney Spleen Duodenum Colon Small Intestine Large Intestine Skin Bone/cartilage Tumor Epithelium Endothelium Cultured cells (stored dry) Cultured cells (fresh) Cytonin TM Pro K (1:50) Pro K (1:200) Mg 2+ Co 2+ Mn 2+ 22

24 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. DAB Solution contains diaminobenzidine, which is harmful if swallowed, inhaled, or absorbed through the skin. Handle and dispose of according to local regulations. 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. Reactivity Data This kit contains diaminobenzidine, DAB, a suspected carcinogen. Wear gloves, eye protection and protective clothing when handling. Dispose of according to local regulations. TACS, TACS-Nuclease and Cytonin are trademarks of Trevigen, Inc /04 23