TACS XL TM In Situ Apoptosis Detection Kit Catalog Number: TA200 DAB 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.
Contents TABLE OF CONTENTS Page PRINCIPLE OF THE ASSAY 2 REAGENTS PROVIDED........................................................... 3 MATERIALS REQUIRED BUT NOT PROVIDED 3 Reagents.................................................................. 3 Equipment 4 Disposables................................................................ 4 PROCEDURES 4 Reagent Preparation......................................................... 4 Preparation of Cells 7 Preparation of Tissues........................................................ 9 Labeling Procedure 12 Counterstaining and Preparation for Viewing...................................... 14 Controls 15 DATA INTERPRETATION......................................................... 16 TROUBLESHOOTING GUIDE 17 APPENDICES................................................................... 19 Reagent and Buffer Composition 19 Fixation Methods........................................................... 19 TACS-Nuclease Treated Control 20 Double Labeling Hints and Tips................................................ 20 Assembling a Humidity Chamber 21 Analysis by Electron Microscopy............................................... 21 WARNINGS 22 Hazardous Ingredients....................................................... 22 Handling Precautions 22 Emergency Exposure Procedures.............................................. 22 MANUFACTURED FOR AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place N.E. (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 379-6580 United States of America E-MAIL: info@rndsystems.com DISTRIBUTED BY: R&D Systems Europe 19 Barton Lane TELEPHONE: (0)1235 529449 Abingdon Science Park FAX: (0)1235 533420 Abingdon, Oxon OX14 3NB E-MAIL: info@rndsystems.co.uk United Kingdom R&D Systems GmbH FREEPHONE: (0)800 909 4455 Borsigstrasse 7 TELEPHONE: (0)6122 90980 65205 Wiesbaden-Nordenstadt FAX: (0)6122 909819 Germany E-MAIL: infogmbh@rndsystems.co.uk
PRINCIPLE OF THE ASSAY Apoptosis is characterized by a number of intracellular phenomena such as membrane blebbing, chromatin condensation and nuclear DNA fragmentation. Detection of DNA fragmentation is a widely accepted method to assay for apoptosis: fragmentation can be visualized by agarose gel electrophoresis following DNA extraction. DNA fragmentation can also be detected in situ by incorporating labeled nucleotides onto the free 3 OH ends of the fragments using terminal deoxynucleotidyl transferase (TdT) enzyme followed by detection of the labeled molecules. This type of assay, often referred to as the TUNEL assay, allows monitoring of apoptosis in cell samples or in tissue sections, providing histological localization of the apoptotic cells. TACS XL is designed specifically for the in situ detection of apoptosis in tissue and cell culture samples immobilized on slides. TACS XL combines all the advantages of TACS TdT kits, in conjunction with a highly sensitive antibody detection system targeting nucleotides incorporated onto the 3 OH ends of the DNA fragments. TACS XL comes complete and includes 2 options of permeabilization reagents: Cytonin, a non-lipophilic detergent optimized for the permeabilization of cells prior to in situ detection of apoptosis, and Proteinase K, an enzymatic reagent optimized to permeabilize tissues. TACS XL also offers flexibility researchers are seeking by providing a choice of staining solution (DAB or TACS Blue Label ) and their respective counterstaining solutions. You have the option of purchasing a DAB or TACS Blue Label kit or choosing a basic kit if your laboratory is already set up for histochemistry. Contact R&D Systems for more information on additional apoptosis detection products for the study of apoptosis and cell death. 2
REAGENTS PROVIDED TA200 TACS XL DAB Component # Component Quantity Storage Conditions 4876-05-01 Cytonin 5 ml 2-8 C 4800-30-01 Proteinase K Solution 30 L -20 C 4810-30-02 10X TdT Labeling Buffer 1 100 ml 2-8 C 4810-30-03 10X TdT Stop Buffer 100 ml 2-8 C 4828-30-04 B-dNTP Mix 30 L -20 C 4810-30-05 TdT Enzyme 30 L -20 C 4800-30-06 Streptavidin-HRP 2 30 L 2-8 C 4828-30-12 Streptavidin Diluent 1 7.5 ml 2-8 C 4828-30-06 Anti-BrdU antibody 30 L 2-8 C 4800-30-15 TACS-Nuclease TM 15 L 2-8 C 4800-30-16 TACS-Nuclease Buffer 1.5 ml 2-8 C 4800-30-07 DAB Solution 3.75 ml -20 C 4828-30-18 Methyl Green Counterstain 50 ml 18-24 C 1 Contains 0.01% thimerosal 2 Contains 0.05% thimerosal MATERIALS REQUIRED BUT NOT PROVIDED Reagents 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 (H 2 O 2 ) Methanol Xylenes Mounting medium 3
Equipment 57 C incubator or slide warmer (for paraffin sections) Refrigerated centrifuge (for cell sample preparation) Adjustable pipettors (0-20 µl, 20-200 µl, 200-1000 µl) 37 C incubator Coplin staining jars Standard light microscope Cryostat Humidity chamber Ice bucket 50 ml and 500 ml graduated cylinders Disposables Glass slides, pre-treated for electrostatic adherence 50 ml tubes 1-200, 200-1000 µl pipette tips Microcentrifuge tubes 1.5 and 10 ml serological pipettes Gloves PROCEDURES Reagent Preparation 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 18-24 C. 2. 3.7% 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. 4
3. Proteinase K Solution* 50 µl of Proteinase K Solution is used per sample. Store on ice. Thaw provided Proteinase K at 18-24 C, 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 to 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. Leave at 18-24 C until use. Remove an aliquot of 50 µl per sample for preparing the Labeling Reaction Mix (Step 7) and place on ice. 7. Labeling Reaction Mix* Thaw B-dNTP Mix at 18-24 C, 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: B-dNTP 1 L TdT Enzyme 1 L 1X TdT Labeling Buffer (step 6) 50 L 5
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. Leave at 18-24 C until use. 9. Antibody Solution 50 µl of Antibody Solution is used per sample. To prepare, dilute: Streptavidin-Diluent 50 L Anti-BrdU 1 L 10. Streptavidin-HRP Solution 50 µl of Streptavidin-HRP Solution is used per sample. To prepare, dilute: Streptavidin-Diluent 50 L Streptavidin-HRP 1 L 11. DAB Solution* 50 ml of DAB Solution is used to process 1-10 slides. Thaw DAB at 37 C, then place at 18-24 C. 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. 12. Methyl Green Methyl Green is ready to 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. 6
13. 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. 14. 100%, 95%, 70% ethanol Either 100% (200 proof) or denatured alcohol (90% ethanol, 5% methanol, 5% isopropanol) may be used. Dilute with dh 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. 15. 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 Sample Preparation and Fixation It is important to read through the instructions for use before preparing tissue or cell samples for labeling. There are key steps that are very important for successful labeling. This section includes instructions for Sample Preparation, in situ Labeling and Viewing. The Assay Protocol for Labeling is in tabulated form and details the steps involved in the labeling reaction and in preparing the sample for viewing. Prior to labeling, the samples must be rehydrated, if necessary, and washed in PBS. The labeling procedure begins with samples in PBS regardless of the fixation and immobilization method. Preparation of Cells Preparation of cells in suspension Cells grown in suspension or prepared from dissociated tissues, can be fixed in solution, then spotted onto pretreated glass microscope slides for processing. This method is quick and easy and requires no special equipment. Cells immobilized onto glass slides can be stored for several months. Method 1. Harvest cell suspension by centrifugation at 500 x g for 5 minutes at 18-24 C. 2. Discard media and resuspend at 1.0 x 10 6 cells/ml in 3.7% Buffered Formaldehyde. Let stand for 10 minutes at 18-24 C. 3. Centrifuge at 500 x g for 5 minutes at 18-24 C and discard fixative. 4. Resuspend at 1.0 x 10 7 cells/ml in 80% ethanol. Samples can be stored at this point, see next page. 7
5. Spot 1.0 x 10 5 cells onto a clean glass microscope slide. Dry for 2 hours on a slide warmer at 45 C. Note: Glass slides pretreated for electrostatic adherence are recommended. Other pretreatments (e.g. gelatin) can cause increased background staining. 6. Immerse slide in 70% ethanol for 10 minutes, then air dry overnight at 18-24 C or dry at 45 C for 2 hours. Samples can be stored at this point, see below. 7. Rehydrate by immersing for 5 minutes each in 100%, 95%, then 70% ethanol. 8. Immerse in 1X PBS for 5 minutes and proceed to Labeling Procedure. Note: Labeling directly after fixation is optimal. Storage: Samples can be stored after steps 4 and 6. Following step 4: Cells can be stored in 80% ethanol at 2-8 C for several weeks. However, signal intensity in positive cells will reduce with time due to loss of small DNA fragments. Following step 6: Store samples at 2-8 C in airtight containers with desiccant. Samples can be stored for several months. After storage, proceed with steps 7 and 8 above. Preparation of Cells in Monolayer For optimal outcomes, cells should be grown on a sterile surface that allows for both fixation and direct labeling, such as sterile chamber slides, cover slips or on slides. Cells that grow in monolayer will lift off the substrate during apoptosis, typically fairly late during the apoptotic process. To ensure the entire population of cells is analyzed, harvest the media and spot the recovered cells onto a slide following the procedure for suspension cells. Sterile Chamber Slides Remove the chamber walls and gasket after fixation. The chamber walls and gasket may be left in place during the labeling reaction if different treatments, e.g. no enzyme and nuclease treatment, are required of adjacent samples on the same slide. Sterile Slides The slides must be sterile and, if necessary, pretreated to ensure cell adhesion. Sterilize microscope slides by autoclaving in a large glass Petri dish. If needed, coat slides with sterile poly-l-lysine or collagen. Place sterile microscope slides in culture vessel directly before plating cells. Sterile Glass Cover Slips Culture cells directly on sterile cover slips that are placed into a 12 or 24 well tissue culture plate. Sterilize cover slips by autoclaving in a large glass Petri dish. If needed, coat cover slips with sterile poly-l-lysine or collagen. Place sterile glass cover slips in wells of tissue culture dishes (12 mm cover slips fit into 24 well tissue culture plates) using fine-tipped sterile forceps. Handle only at edges prior to cell plating. Method: 1. Remove media from cells and rinse once with 1X PBS at 18-24 C. 2. Fix cells for 10 minutes at 18-24 C in 3.7% Buffered Formaldehyde. 3. Wash cells one time in 1X PBS. Samples can be stored at this point, see next page. 4. Proceed to Labeling Procedure. 8
Storage: Labeling directly after fixation is optimal. Note: The labeling of some samples is less efficient after storage. Empirical testing is required to determine if samples label efficiently after storage. Two storage methods for fixed samples are provided to store cells after step 3 (previous page): The fixed and washed cells can be stored for up to 1 week in Cytonin at 2-8 C. The samples must be covered to prevent contamination and evaporation. If experimental design dictates a time course extending over several days, storage in Cytonin is recommended. After storage, proceed with step 3 of Labeling Procedure. The fixed cells can be dehydrated for storage. Dehydrate by immersing in 70%, 80%, 95% and 100% ethanol for 5 minutes each followed by air drying for 10 minutes each. Samples may then be stored at 2-8 C with desiccant for several months. After storage, rehydrate by immersing for 5 minutes each in 100%, 95% and then 70% ethanol. Immerse in 1X PBS for 5 minutes and proceed to Labeling Procedure. Preparation of Tissues Use of glass slides pretreated for electrostatic adherence is recommended for all tissues. Preparation of Fresh Unfixed Tissue Fresh tissue requires minimal fixation and frozen samples are easily permeabilized for labeling. Some disadvantages include the difficulty in collecting good quality sections, the need to cut thicker sections, and poor retention of morphology. Frozen sections are less resistant to protease treatments and can lift off if not collected onto the appropriately pretreated slides and dried thoroughly. Samples must be fixed prior to labeling. Freeze Tissue Rapid freeze tissue or biopsy immediately after removal by immersing in liquid nitrogen or on dry ice. Samples may be embedded in a cutting matrix. Position the sample within cutting matrix in a suitable container. Immerse embedded tissue in isopentane chilled on dry ice. Frozen samples may be stored for many months at -80 C. Cryosection Frozen Tissue Using the cutting matrix, attach the sample to cutting block and equilibrate to the temperature of the cryostat before sectioning. Collect sections between 6-15 µm on glass slides pretreated for electrostatic adherence. Helpful Tips for Cryosectioning: Individual expertise and tissue type will determine the thickness of the sections. Sections between 10-15 µm provide the best results. Sections between 6-9 µm tend to tear during cutting, resulting in rough edges that can increase the background. Up to 3 sections per slide can be placed; each spaced well apart. The spacing prevents reagent mixing between samples. 9
Method after Sectioning: 1. It is critical to dry the samples thoroughly after sectioning. Dry overnight at 18-24 C or for at least 2 hours at 45 C on a slide warmer. Samples can be stored at this point, see below. 2. Rehydrate by immersing for 5 minutes each in 100%, 95%, then 70% ethanol. 3. Wash once in 1X PBS for 5 minutes. 4. Fix samples by immersing in 3.7% Buffered Formaldehyde for 10 minutes at 18-24 C. 5. Wash cells once in 1X PBS for 5 minutes. 6. Proceed to Labeling Procedure. Note: Labeling directly after sectioning is optimal. Storage: Store slides at -80 C with desiccant, for up to 3 months. After storage, equilibrate samples to 18-24 C and redry for 2 hours at 18-24 C or on a slide warmer at 45 C. Proceed with method as indicated above starting at Step 2. Preparation of Fixed Samples - Immersion or Perfusion Samples are routinely fixed by immersion or perfusion methods. After fixation, samples are cryosectioned or paraffin-embedded. Fixation Methods Immersion Fixation The fixation time should ensure good cross-linking but prevent tissue from becoming hard and brittle. Some empirical determination of the optimal fixation time may be required. Immerse relatively small pieces of tissue (1 cm 3 ) in at least 10 volumes of 3.7% Buffered Formaldehyde. After 30 minutes, change to fresh fixative and leave at 18-24 C up to 24 hours. Helpful Tips: Tissues with high cellularity require longer fixation times; other tissues are often fixed within 2 hours. If alternative fixatives are used, it is recommended that a pilot study be performed to ensure that the fixative allows for permeabilization and labeling. Alternative immersion fixatives include paraformaldehyde, glutaraldehyde or Bouin s fixative. Perfusion Fixation Standard laboratory procedures should be followed for perfusion fixation. Formaldehyde, paraformaldehyde or glutaraldehyde may all be used as fixatives. After 2 hours of perfusion, the dissected tissue should be immersed in fresh fixative for up to 24 hours. Storage of Fixed Samples Fixed samples may be stored for long periods. For long term storage, use 70% ethanol or sterile 1X PBS at 2-8 C to avoid extended exposure to fixative. Material that has been stored in fixative for months or years will be more difficult to permeabilize and may not be useful for in situ detection of apoptosis due to DNA hydrolysis during storage. 10
Sectioning Methods Cryosection Fixed Tissues Immersion of tissues in sucrose solution does not affect labeling. Use high grade sucrose prepared in DNase free water. Collect sections of 6-10 µm onto slides pretreated for electrostatic adherence of samples and dried as described in section Preparation of Fresh Unfixed Tissues. Alternatively, sections can be collected directly into 1X PBS and the labeling performed on floating sections. Note: When collecting onto slides from buffer, use either a low salt buffer or dh 2 O to ensure that samples adhere to slides. Storage of Sectioned Tissues Samples may be stored after sectioning, with desiccant at -80 C for up to one month. After storage, the slides should be equilibrated to 18-24 C and dried for 2 hours at 18-24 C or at 45 C on a slide warmer. Rehydrate samples before labeling by immersing for 5 minutes each in 100%, 95%, then 70% ethanol and immerse slides in 1X PBS for 10 minutes. Proceed to Labeling Procedure. Section Paraffin-Embedded Tissues Paraffin-embedding is a routine procedure in many laboratories and is commonly performed by automated equipment. Optimal labeling is achieved when the samples are processed within days of sectioning. Sections between 3-6 µm should be collected onto slides pretreated for electrostatic adherence. Note: The temperature of the molten paraffin must not exceed 65 C, otherwise additional DNA damage can occur leading to spurious positives and high background staining. Prior to the labeling reaction, the samples must be deparaffinized. Deparaffinization Method 1. Immerse sections in two changes of xylenes, 5 minutes each. 2. Immerse sections in 100%, 95%, then 70% ethanol, 5 minutes each. 3. Wash 2 times in 1X PBS, 5 minutes each. 4. Proceed to Labeling Procedure. Storage It is preferable to store the uncut paraffin block at 18-24 C, as opposed to the sections. The xylenes and ethanols used for deparaffinization can be reused several times (up to 100 slides may be processed in 50 ml), but they must not be used for rehydration of non-embedded samples or for dehydration after performing the labeling reaction. 11
Labeling Procedure Note: Details on the Labeling Procedure are provided in the table following these descriptions. Labeling Samples on Slides: Wash slides using small Coplin histology jars. Each jar holds up to 50 ml of buffer and up to 10 slides. For procedural steps involving 50 µl per sample, place slides on a flat surface and spot reagent from above using a pipette tip; do not touch the sample. Small biopsy samples are easily covered with 50 µl. If 50 µl does not cover the sample, cover slips should be used after pipetting the 50 µl volume. Slowly lower the cover slip from one edge to prevent formation of any air bubbles. If bubbles are trapped, remove cover slips by dipping the slide vertically in dh 2 O and reapply new reagent and cover slip. Labeling Samples in Chamber Slides: Remove chamber walls and gasket after fixation and process as described for slides. Use cover slips for all steps involving 50 µl reaction volumes. If different labeling reactions are performed on samples on the same slide, leave the plastic walls in place until after the labeling reaction, then remove the plastic walls and rubber gasket and proceed as described above. Labeling Samples on Glass Cover Slips: Process the 12 mm glass cover slips with the cell-side facing up in the 24 well tissue culture plate. Wash by filling the wells with buffer and removing with a Pasteur pipette (use a gentle vacuum if available). Spot the 50 µl reaction volumes directly onto the cover slip. Alternatively, spot the 50 µl reaction buffers onto a clean glass slide, then remove 12 mm glass cover slip from the well and flip it over, cell-side down, on top of the reagent. Use fine-tipped forceps and handle glass cover slips only at the very edges. For dehydration and clarification, dip the 12 mm glass cover slips individually in ethanol series and xylenes for 20 seconds. Note: Xylenes will melt plastics, therefore, do not add xylenes to the tissue culture plates. 12
Labeling Protocol: Note: If samples have not been rehydrated, proceed with step 1. If samples have been rehydrated, continue with step 2. 1. 2. Rehydrate samples in decreasing alcohol series for 5 minutes each. Then immerse samples in 1X PBS for 5 minutes at 18-24 C. Carefully dry glass slide around sample. Cover sample with 50 L of Proteinase K Solution and incubate 5-30 minutes at 18-24 C. OR Cover sample with 50 L of Cytonin and incubate 15-30 minutes at 18-24 C. If necessary, use cover slips. Do NOT allow samples to dry. Samples must be rehydrated immediately before labeling. Use Proteinase K for most tissue samples. Use Cytonin for most cell samples. Some empirical determination may be required. It is recommended that Cell Culture Control Slides (Cat. # 4800-30-21) be treated with Proteinase K Solution for 15 minutes. 3. Wash 2 times in dh 2 O, 2 minutes each. For Nuclease-treated samples, wash in dh 2 O before using TACS-Nuclease. Refer to the appendix. 4. 5. 6. 7. 8. 9. 10. 11. 12. Immerse slides in Quenching Solution for 5 minutes at 18-24 C. Wash samples in 1X PBS for 1 minute at 18-24 C Immerse slides in 1X TdT Labeling Buffer for 5 minutes at 18-24 C. Cover sample with 50 L of Labeling Reaction Mix and incubate at 37 C for 1 hour in a humidity chamber. If necessary, use cover slips. Immerse samples in 1X TdT Stop Buffer for 5 minutes at 18-24 C to stop labeling reaction. Wash samples 2 times in 1X PBS for 5 minutes each at 18-24 C. Cover sample with 50 L of diluted anti-brdu and incubate for 1 hour at 37 C. Wash samples 3 times in 1X PBS, 0.05% Tween 20 for 2 minutes each. Cover sample with 50 L of Streptavidin-HRP Solution and incubate for 10 minutes at 18-24 C. Refer to Reagent Preparation. Do not leave longer than 5 minutes since H 2 O 2 can damage DNA. Refer to Reagent Preparation. Refer to Reagent Preparation. Use a humidity chamber during incubation time (see Appendix). Labeling can be reduced to 30 minutes. Refer to Reagent Preparation. For details on a humidity chamber, see the Appendix. Refer to Reagent Preparation. Refer to Reagent Preparation. Refer to Reagent Preparation. 13. Repeat step 11. 14. Immerse samples in DAB Solution for 2-7minutes. Use caution when handling DAB Solution. Refer to Reagent Preparation. 15. 16. Wash samples in several changes of deionized water, 2 minutes each. Proceed to counterstaining and preparation for viewing. 13
Counterstaining and Preparation for Viewing Method A - For most cells and tissues 1. Immerse samples in deionized H 2 O for 2 minutes. 2. Immerse samples for 5 seconds to 5 minutes in Methyl Green. 3. Wash slides sequentially by dipping at least 10 times each in: dh 2 O 70% ethanol, 2 changes 95% ethanol, 2 changes 100% ethanol, 2 changes Xylenes, 2 changes 4. Wipe off excess xylene from the back of the slide and lay slide flat. 5. Place 1 drop, about 50 µl, of mounting media onto sample. 6. Lower glass cover slip onto sample and apply gentle, even pressure to expel air bubbles. 7. Leave slide flat overnight to allow mounting media to harden. 8. Store slides in the dark. Method B - Use as an alternative to Method A 1. Immerse samples in deionized H 2 O for 2 minutes. 2. Immerse samples for 5 seconds to 5 minutes in Methyl Green. 3. Wash slides sequentially by dipping 10 times each in: 1-Butanol, 1-Butanol, until sample turns from blue to green Xylene, 2 times (5-10 seconds each) 4. Wipe off excess xylene from the back of the slide and lay slide flat. 5. Place 1 drop, about 50 µl, of mounting media onto sample. 6. Lower glass cover slip onto sample and apply gentle, even pressure to expel air bubbles. 7. Leave slide flat overnight to allow mounting media to harden. 8. Store slides in the dark. Cells and tissues may be counterstained with Methyl Green. Glass cover slips can be held with fine-tipped forceps and dipped individually into the stains and ethanols. Spot only 25 µl mounting media onto a clean glass slide and mount the cover slip (cell side down) onto the slide. If a plastic support was used for cell culture, do not pass through xylenes. An aqueous mounting media can be placed on the sample and results viewed directly. Note: Follow manufacturer s instructions. 14
Controls TACS Nuclease-treated Control After incubation in Cytonin or Proteinase K Solution and washing in water, treat one sample with TACS-Nuclease to generate DNA breaks in every cell. 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 brown nuclear staining when stained with DAB. Refer to the Appendix for the procedure. Additional experimental controls that may be included when performing the protocol on new samples are listed below: Unlabeled Experimental Control Sample The TdT Enzyme should be omitted from the Labeling Reaction Mix for one sample. This control will indicate the level of background labeling associated with non-specific binding of the streptavidin-hrp. This control should not have any brown staining. Experimental Negative Control Sample 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 an 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 may stain positively with the DAB. Counterstaining Controls Although uncommon, some cells and tissues may take up excessive amounts of Methyl Green, obscuring specific 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 2-30 seconds have been noted. It is recommended to start with a 5 second immersion incubation in counterstain and alter accordingly. Apoptotic Cells Control Cell Culture Control Slides (Cat. # 4800-30-21) can also be used. Each control slide contains both positive and negative cells for apoptosis. Area 1 contains apoptotic cells, Area 2 contains untreated cells and Area 3 is blank. 15
DATA INTERPRETATION Apoptosis is defined by morphological criteria. The morphological data obtained from standard microscopy and histochemistry should be considered in conjunction with the biochemical assays used 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 blue staining that is not associated with nuclei. The suggested experimental controls are important in data interpretation. The controls allow optimization of in situ detection of apoptosis without expending valuable test samples. Under optimal conditions, the Unlabeled Control (i.e. enzyme omitted) should show no brown staining, the TACS Nuclease-treated sample should show pale brown staining in almost all cells, and the Experimental Negative Control should have less than 20% brown-stained cells. The Nuclease-treated samples allow confirmation that the reaction was performed correctly. The brown staining of 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 results. Cell Culture Control Slides (not included in the kit) are used to demonstrate apoptotic cell morphology that can be expected in cell culture. In Area 1, cells may be unevenly counterstained with some cells taking up more counterstain, particularly condensed cells (a majority of cells will be positive for DNA fragmentation indicated by a brown stain). In Area 2, cells should have less than 5% positively-stained cells. The shape and size of these cells will be more uniform and should be evenly counterstained. 16
TROUBLESHOOTING GUIDE Problem Possible Cause Solution Brown staining of cells when the TdT Enzyme is omitted from the Labeling Reaction Mix. No staining in Nuclease-treated sample. Endogenous peroxidase activity inadequately quenched. Excessive peroxidase activity in sample (rare). Non-specific binding of Streptavidin-HRP solution. Sample dried out during the labeling procedure. Poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue) preventing enzyme access. No DNA left in sample due to hydrolysis (poor storage of samples or sections). Excessive (removed all DNA) or inadequate Nuclease treatment. TdT Enzyme is inactive. The enzyme is the most labile component in the kit. Color Development reaction failed. Use fresh aliquots of 30% H 2 O 2. Increase concentration of H 2 O 2 in Quenching Solution to 5%. Increase number of washes after binding. Prepare Streptavidin-HRP Solution in 1X PBS, 1% BSA. Decrease concentration of Streptavidin-HRP by diluting stock solution up to 1:200. Use cover slips (or lids for plates or chamber slides) and incubate in a humidity chamber. Increase incubation time with Cytonin. Samples can be stored for up to 1 week in Cytonin at 2-8 C. Refer to suggestions for storage. Optimize time for Nuclease treatment (5 minutes up to 2 hours). TdT Enzyme must be stored at -20 C (not a frost-free freezer). Do not bring enzyme up to ice temperature. Place in a -20 C freezer block or remove an aliquot from the tube directly in the freezer. Use freshly prepared Quenching Solution made from fresh 30% H 2 O 2. To check reaction, spot 1 L of Streptavidin-HRP onto 1 cm 2 of 3M paper and air dry. Immerse the filter paper in DAB Solution to check for brown coloration compared with a piece of plain paper. 17
Problem Possible Cause Solution Labeling of majority of cells in the Negative Experimental Control (e.g. Normal tissue or untreated cells) when there is no labeling if the enzyme is omitted and satisfactory labeling of the Nuclease-Treated control. No labeling in experimental sample. Experimental sample shows extensive cytoplasmic staining. Methyl Green counterstain is dark blue. High level of apoptosis (or necrosis) in Negative Control. Prolonged incubation with DAB leads to precipitate over the entire sample. Excessive treatment with Proteinase K Solution. No apoptosis (or necrosis) occurring in sample. High rate of cell death, late apoptosis or necrosis. Overstaining. Select a more appropriate Negative Control or inhibit apoptosis in cell culture (e.g. with protein synthesis inhibitors). Check morphology of cells prior to assay for evidence of excessive apoptosis. Reduce time of DAB treatment. Dilute Proteinase K to 1:200. If all controls gave the expected results and were processed at the same time as the experimental sample, there may be no DNA fragmentation in cells within the sample. Necrotic samples will exhibit cytoplasmic staining. Reduce time or intensity of treatment. Apoptosis in cell culture will progress to necrosis. Reduce time of treatment in cell culture. Reduce time in Methyl Green. Increase the number of washes in 95% ethanol or butanol. 18
APPENDICES Reagent and Buffer Composition 10X PBS, ph 7.4 25 mm sodium dihydrogen phosphate (NaH 2 PO 4 ) 75 mm disodium hydrogen phosphate (Na 2 HPO 4 ) 1.45 M sodium chloride (NaCl) Deionized, sterile water Note: Water used should be DNase free Distilled autoclaved water can be used Cytonin Proprietary permeabilization reagent TACS-Nuclease Proprietary endonuclease TACS-Nuclease Buffer 50 mm Tris-HCl, ph 8.0 1 mm MgCl 2 100 µg/ml BSA 10X TdT Labeling Buffer 1 M TACS Safe-TdT Buffer 0.5 mg/ml BSA (RIA grade) 0.6 mm 2-mercaptoethane sulfonic acid (MESNA) 10X TdT Stop Buffer 0.1 M EDTA, ph 8.0 B-dNTP Mix Optimized nucleotide mix of brominated dntps Anti-BrdU antibody Biotinylated mouse monoclonal anti-bromodeoxyuridine Streptavidin-Diluent Optimized blocking reagent Methyl Green 1% Methyl Green Fixation Methods There are several fixation methods commonly used that are appropriate for the protocol described in the instructions for use. Formaldehyde is the recommended fixative based on laboratory testing. However, other fixatives that maintain DNA integrity may be used. These include paraformaldehyde and glutaraldehyde. Alcohol fixatives such as ethanol, methanol or acetone are not recommended. Regardless of the fixative used, it is important not to fix cells and tissues for extended periods of times. Fixation method will likely be dictated by immunocytochemistry protocols in double-labeling experiments (see next page). Post-fixation in acetone, ethanol or methanol is common in preparation of tissues and is usually compatible with TACS XL. Any alternative fixation method used should be tested in a pilot labeling study to ensure compatibility with TACS XL. 19
TACS-Nuclease Treated Control Treat one sample with TACS-Nuclease to generate DNA breaks in the majority of cells. Insert the Nuclease treatment step after permeabilization of the cells (step 2 of the Labeling Procedure). 1. Wash sample in dh 2 O a. 2. Prepare Nuclease solution (50 µl per sample): TACS-Nuclease Buffer 50 L TACS-Nuclease 1 L 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 dh2 O before using TACS-Nuclease. b The length of incubation with the Nuclease varies in function of 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). Double Labeling Hints and Tips The in situ labeling protocol described here is useful for double labeling experiments when the occurrence of apoptosis can be correlated with cellular antigens against which antibodies are available. Note: The antibody 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 provided in the Appendix (see previous page). Post-treatments used in immunocytochemistry to permeabilize or expose antigenic determinants include treatment with proteases, acid or base, detergent and/or microwaving. Permeabilization with Cytonin may be sufficient for many antibodies and additional treatment may not be needed. Protease treatment is not recommended on most samples because samples will often disintegrate later during immunocytochemistry or DNA labeling. Strong acid or base treatment should also 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. 20
Empirical determination of optimal conditions for immunohistochemistry and in situ detection of apoptosis must be completed in separate experiments first. Combine the two methodologies only after optimizing them separately on identical 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. Many options are available for double labeling experiments. If the antigen is nuclear, carefully select the detection label and appropriate 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; for example TACS Blue Label cannot be used with Blue counterstain. 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. TACS Blue Label 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 TACS Red Label TM (Cat. # 4825-30-RL). 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 tissue. 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. Analysis by Electron Microscopy The protocol given here can be adapted for electron microscopy. Both pre- and post-embedding labeling can be performed depending upon 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 BrdU, use the biotinylated anti-brdu and a streptavidin conjugated to colloidal gold. Incubate overnight at 2-8 C. Wash and stain with uranyl acetate. For some samples, embedding after labeling 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. 21
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 XL, TACS, TACS-Nuclease, TACS Blue Label, TACS Red Label, and Cytonin are trademarks of Trevigen, Inc. Tween is a registered trademark of ICI Americas. 22
NOTES 04.99 750405.8 5/02 23