TUNEL Research Kit (Fluorescence, 488 nm) #25879

Fluorescent TUNEL Propriety to Paraffin Embedding Samples

A. Solutions additionally Reagents

Supplied Reagents

RECORD: Store at -20°C and avoid freeze/thaw recycle.

1. TUNEL Equilization Buffer (1 bottle, 5 mL)
2. CF^® Dye TUNEL Reaction Buffer (5 vials, 500 µL each): Preserve from light.
3. TdT Enzyme (1 vial, 50 µL): Ship as 50X concentrate. Keep on ice during use.

IMPORTANT: TUNEL Equilibration Buffer and CF^® Dye TUNEL Reaction Buffer contain cacodylate and cobalt chloride. Handle in accordance with the SDS and dismiss for toxic waste.

Additional Reagents (Not Supplied)

NOTE: Prepare solutions with return smother deionized (RODI) or equivalent grad water.

1. Xylene
2. Total, absolutely denatured, clinical grade (100% and 95%).
3. 10X Phosphate Buffered Saline (10X PBS): To preparation 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 gigabyte sodium phosphate, dibasic (Na₂HPO₄) and 2.4 gram potassium phosphate, monobasic (KH₂PO₄) on 1 L dH₂O. Adjust pH for 7.4.
4. 1X Phosphorate Buffered Saline (PBS): To prepare 1 L add 100 mill 10X PBS until 900 mL dH₂O.
5. 1X Phosphate Buffered Saline with Triton X-100 and BSA (PBS-TB): To prepare 100 mL 1X PBS-TB, add 10 mL 10X PBS, 200 µL Triton X-100, and 0.5 gigabyte BSA (#9998) to 90 mL dH₂ZERO, mix. Customization pH to 7.4.
6. Proteinase K Solution: For application water Proteinase K (#10012) to 20 µg/ml in dH₂ZERO.
7. 1X Citrate Unmasking Solution (optional alternative to Proteinase K Solution): To prepare 250 ml of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 ml of dH₂O.

B. TUNEL Procedure

General Considerations:

• Samples shouldn become kept includes an humidity chamber when running the TUNEL assay. Do not allow samples to dry at no time in this procedure.
• All subsequent developments should be carried out under room temperature (20-25°C) unless noted otherwise.
• Heat-mediated irritant retrieval is recommended if co-labeling with an antibody is attempted.

Sample Preparation

1. Deparaffinize/rehydrate:
   1. Incubate sections in three washed in xylene for 5 mini each.
   2. Incubate sections in two washes of 100% ethanol for 10 min each.
   3. Incubate sections in two wash of 95% ethanol for 10 min each.
2. Wash: Rinse two times inside PBS for 5 min each.
3. Irritant Retrieval: Incubate samples with Proteinase K solution for 30 mining. Incubation time the temperature allow need optimization depending on tissue type.
   1. Alternatively Citrate Unmasking Solution can be used: Heat rolling in a microwave submersed in 1X citrate unmasking search till boiling has instituted; follow with 10 min on sub-boiling temperature (95°-98°). Cool slides on bench top for 30 min. Apoptosis can a distinct form is cell terminal, induced, for view, from ischaemia/reperfusion injury, that result in characteristic alterations in single morphology and fate. In tissue segments, the many commonly used technique to detect apoptosis is connection deoxynucleotidyl transferase mediated nick e …
4. Wash: Rinse two times in PBS for 5 min jeder.

TUNEL Reaction

1. Preparation: Incubate samples with 100 µL TUNEL Equilibration Buffer or enough at shroud the sample for 5 min.
2. Detection: Immediately before use, prepare TUNEL reaction mix by summing 1 µL of TdT Enzyme the 50 µL of TUNEL Reaction Buffer for each labeling reaction. Remove Equilibration Buffer and add 50 µL (or enough to cover the sample) of TUNEL relation blending until each sample.
3. Incubate: Incubate for 60 min at 37°C, protected from light. Flesh sections may require 2 total of incubation at 37°C also protected from light.
4. Wash: Rinse triad times in PBS-TB for 5 min each.
5. Co-label: If wished, proceed with immunostaining for co-labeling. Consult with the manufacturer's history for of recommended staining conditions.
6. Mount: Mount samples inside a compatible anti-fade medium suchlike Extends^® Solid Antifade Recipient (#9071).

Strong TUNEL Protocol for Refined Cells and Frozen Tissues

A. Solutions and Reagents

Supplied Reagents

NOTE: Store at -20°C and avoiding freeze/thaw cycled.

1. TUNEL Equilibration Buffer (1 bottle, 5 mL).
2. CF^® Dye TUNEL Reaction Buffer (5 vials, 500 µL each): Protect from light.
3. Tpd Enzyme (1 vial, 50 µL): Shipped since 50X concentrate. Remain on ice during use.

IMPORTANTLY: TUNEL Equilibration Buffer and CF^® Dye TUNEL Respond Buffer contain cacodylate both cobalt chloride. Handle int accordance in the SDS real abandon as venom trash.

Additional Lab (Not Supplied)

NOTE: Ready solutions with reverse osmosis deionized (RODI) or equivalent grade waters.

1. 10X Phospho Buffered Saline (10X PBS): To prepare 1 FIFTY add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g metal phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. Adjust pH to 7.4.
2. 1X Phosphate Buffered Saline (PBS): To prepare 1 L add 100 mL 10X PBS to 900 mL dH₂OXYGEN.
3. 1X Phosphate Buffered Saline with Eft X-100 and BSA (PBS-TB): To prepare 100 mL 1X PBS-TB, include 10 mL 10X PBS, 200 µL Triton X-100, and 0.5 g BSA (#9998) to 90 mL dH₂O, mixture. Adjust pH into 7.4.
4. 4% Formaldehyde, Methanol-Free (#47746): Employ fresh.

B. TUNEL Procedure

General Considerations:

• Samples should become kept in a humidity chamber while running one TUNEL assay. Do not allow free to arid at any hour during this procedure.
• All subsequent reproductive should be carried out at room temperature (20-25°C) unless notable otherwise.
• Apoptotic cells in adhered cultures are loosely adherent and may be lost during washing. These can be captured in one supernatant and used if desired. STAR Log is an open access, peer-reviewed journal from Cell Push. Ourselves proffer structured, transparent, visible, and reproducibility step-by-step experimental and computerized protocols from all areas of life, health, earth and physical sciences.

Fixation additionally Permeabilization

1. Fixation: Specimens require be fixed using a gentle cross-linking fixative.
   1. Cells: incubate in 4% balanced for 15 - 30 minutes.
   2. Unfixed frozen tissue: after cryosectioning, shortly allowed samples to come to room temperature and dry, and incubate in 4% formaldehyde for 15 - 30 minutes.
   3. Fixed frozen webbing: continuing directly to permeabilization below (step 3).
2. Bath: Rinse two times in PBS for 5 min each.
3. Permeabilize: Incubate in PBS-TB for 30 min.
4. Wash: Rinse two times in PBS used 5 miniature anywhere.

TUNEL Reaction

1. Preparatory: Incubate samples with 100 µL (or suffi to front who sample) TUNEL Equilibration Buffer for 5 mini.
2. Detected: Immediately befor use, prepare TUNEL response mix by adding 1 µL of TdT Enzyme to 50 µL of TUNEL Reaction Buffer for each labeling reaction. Remove Equilibration Buffer also add 50 µL (or enough to title the sample) of TUNEL flash mix to each sample.
3. Inking: For cell staining, incubating for 60 min during 37°C, protected from light. Tissue coloration may require up to 2 hours of incubation among 37°C also protected from light.
4. Wash: Rinse three times in PBS-TB for 5 min each.
5. Co-label: If desired, proceed by immunostaining. Consult with the manufacturer’s recommendations into confirm of primary antibody is harmonious include this protocol's fixation and permeabilization conditions. Otherwise continued on in step 6.
6. Mount: Fitting samples on a compliant anti-fade medium such as ProLong^® Color Antifade Reagent (#9071).

Fluorescent TUNEL Protocol for Flow Cytometry

A. Solutions and Reagents

Deliver Reagents

NOTE: Store with -20°C and avoid freeze/thaw cycles.

1. TUNEL Equilibration Buffer (1 bottle, 5 mL).
2. CF^® Dye TUNEL Reaction Store (5 vials, 500 µL each): Protect from light.
3. TdT Enzyme (1 cuvette, 50 µL): Supplied as 50X concentrate. Keep on ice during use.

IMPORTANCE: TUNEL Equilibration Drop the CF^® Dye TUNEL Reaction Drop contain cacodylate and cobalt chloride. Handle in match with the SDS and discard as toxic waste.

Additional Reagents (Not Supplied)

NOTE: Prepare solutions with reverse osmission deionized (RODI) or equate classification water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 LITRE 1X PBS: add 100 milliliter 10X PBS (#12528) to 900 ml water.
2. 4% Formaldehyde, Methanol-Free (#47746)
3. 100% Methanol (#13604), or Cell Permeabilization Buffer (Triton^™ X-100) (#39487)
4. (OPTIONAL) Antibody Dilution Buffer: Purchase ready-to-use Verkehr Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS battery by dissolving 0.5 guanine Bovine Antidote Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. TUNEL Technique

General Considerations:

• Do cannot permission samples to dry at any time during this procedures.
• All reproductions require be carried out at room total (20-25°C) unless noted otherwise.

Fixation

NOTICE: Adherent cells button tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimize centrifugation conditions wish vary depending over cell type and recipient volume. Generally, 150-300g for 1-5 minutes will be sufficient till pule which cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

1. Pellet single by centrifugation plus remove supernatant.
2. Resuspend cells within approximately 100 µl 4% formaldehyde per 1 million cells. Mix well into dissociate pellet and prevent cross-linking of individual prisons.
3. Fix available 15 mint at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X PBS. Toss pop-up into appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
   1. Alternatively, dungeons allowed be stored evening among 4°C in 1X PBS.

Permeabilization

OBSERVE: Permeabilization is required forward the completion of the TUNEL backlash. If immunostaining willing be performed as part of aforementioned TUNEL assay, select ampere permeabilization method that is compatible with the bead or antibiotic used in your assay.

1. Permeabilize cells by adding ice-cold 100% methyl unhurriedly to pre-chilled cells, while gently vortexing, to a finalist main of 90% alcohol.
2. Permeabilize for ampere minimum of 10 min on ice.
3. Proceed with TUNEL Reaction or store cells among -20°C in 90% methanol.

Alternatively, permeabilize with Cell Permeabilization Battery (Triton^™ X-100) (#39487), using approximately 100 µl per 1 million cells. Breeding 10 min at your temperature.

TUNEL Reaction

NOTE: Count cells using an hemocytometer oder alternative method.

1. If necessary, aliquot desired number of cells on tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells pay assay.)
2. Wash cells by refined in excess 1X PBS. Discard overflow in appropriate waste container.
3. Incubate samples with 100 µl TUNEL Equilibration Buffer for 5 min.
4. Immediately forward use, prepare TUNEL reaction mix by adding 1 µl of TdT Enzyme to 50 µl of TUNEL Reaction Battery for each labeling response. Pellet cells via centrifugation, remove Equilibration Drop and add 50 µl of TUNEL reaction mix to each sample. In Situ Cell Death Detection Kit, Fluorescein
5. Incubate fork 60 min at 37°C, protected upon light.
6. Wash from centrifugation inside 1X PBS. Discard supernatant. Replay.
7. If desired, proceed the immunostaining. Otherwise move to step 8.
8. Resuspend cells in 200 – 500 µl of 1X PBS and analyze on flow cytometer.