In this protocol, we will discuss how to label cell proliferation in vivo with Invitrogen Click-iT EdU Imaging Kit. Click-iT cell proliferation assays can be used in vivo following EdU administration. EdU is cell permeable and can be delivered in vivo through methods including injection (intraperitoneal, intramuscular, subcutaneous), in drinking water, or direct incubation in certain organisms (e.g., Drosophila and zebrafish larvae) where EdU becomes incorporated into actively dividing cells. The Click-iT technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling. Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period for easy quantitation.
This protocol can be used for:
- Detecting DNA synthesis using a fluorescence microscope
This protocol should not be used for:
- Flow cytometry; for a flow cytometry protocol, see Click-iT EdU Protocol for Flow Cytometry
You will need the following for this protocol:
Reagents
- Invitrogen Image-iT Fixative Solutions 4% Formaldehyde Fixative Solution in PBS (methanol-free) (Cat. No. R37814)
- Thermo Scientific Chemicals Xylenes, 99%, for biochemistry and histology (Cat. No. 447240010)
- Gibco Phosphate buffered saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
- Thermo Scientific Absolute Ethanol, 200 proof, Molecular Biology Grade (Cat. No. T038181000CS)
- Invitrogen EdU (5-ethynyl-2’-deoxyuridine) (Cat. No. E10187)
- Click-iT EdU Imaging Kit (Cat. Nos. C10338, C10340, C10337, C10339)
- Invitrogen Image-iT Fixation/Permeabilization Kit (e.g., 0.5% Triton X-100 in PBS, Cat. No. R37602)
- 3% bovine serum albumin (BSA) in PBS (3% BSA in PBS), pH 7.4
- For dual pulse 5-Bromo-2'-deoxyuridine (BrdU), 99%, Thermo Scientific Chemicals (Cat. No. H27260.06)
- Invitrogen BrdU Monoclonal Antibody (MoBU-1) (e.g., Cat. No. B35128)
Protocol
EdU labeling in FFPE tissue
Table 1. Examples of EdU concentrations and incorporation into animal model systems.
Species | Cell or Tissue type | Cell or Tissue Preparation | EdU Conc | EdU incubation time | Administration method | Detection | Reference (link) |
---|---|---|---|---|---|---|---|
Mouse | Colon | Frozen, fixed tissue | 1 mg/mouse | 4–144 hours | IP | Alexa Fluor 488 | J Immunol 188(5):2427-2436 (2012). |
Small intestine and brain | FFPE (brain and small intestine), fresh, fixed (small intestine) tissue | 100–200 μg | 24–96 hours | IP | Alexa Fluor 568 and TMR | Proc Natl Acad Sci U S A 105(7):2415-2420 (2008). | |
Retina (dissociated cells) | Fixed cells | 25 μg/g | 4 hours | IP | Alexa Fluor 647 | Mol Biol Cell 23(22):4362-72 (2012). | |
Embryonic brain | Cryosection tissue | 5 µg/g | 24–72 hours | IP (pregnant dams) | Alexa Fluor 594 | Proc Natl Acad Sci U S A 19;110(8):3113-8 (2013). | |
organ of Corti (cochlear neurosensory epithelia) | Whole mount tissue | 50 mg/kg | 4 hours | Subcutaneous injection | Alexa Fluor 488 | J Neurosci 31(24):8883-93 (2011). | |
Optic nerves | Cryosection, fixed tissue | 0.2 mg/mL | 2–8 weeks (water exchanged every 72 hours) | Drinking water | Alexa Fluor 594 | J Neurosci 32(36):12528-42 (2012). | |
Rat | Lung | Cryosection, fixed tissue | 50 mg/kg | 24 hours | IP | Alexa Fluor 488 | Anal Cell Pathol (Amst) 2015:326385 (2015). |
Olfactory Epithelium | Cryosection, fixed tissue | 50 mg/kg | 2 hours | Subcutaneous | Alexa Fluor 594 | J Neurosci 38(21):5022-5037 (2018). | |
Bone (HSPCs from bone marrow) | Fresh and fixed cells | 60 mg/kg | 24 hours | IP (pregnant dams) | Alexa Fluor 647, Alexa Fluor 488 for flow | Nat Commun 13(1):1327 (2022). | |
Zebrafish | Larval intestine | FFPE tissue | 100 μg/mL | 16 hours | Soaking | Alexa Fluor 488 | Proc Natl Acad Sci U S A 108 Suppl 1(Suppl 1):4570-4577 (2011). |
Larval jaws | Fixed tissue | 400 μM | 24 hours | Soaking | Click-iT EdU | FASEB J 35(11):e22002 (2021). | |
Cardiac tissue | Cryosection, fixed tissue | 10 mM | 3 days | IP | Click-iT EdU | Elife 10:e66079 (2021). | |
Drosophila | Larval brain, eye discs & trachea | Fixed tissue | 20–100 μM | 10–60 minutes | Soaking | Alexa Fluor 594 | Development 138(23):5201-5212 (2011). |
Larval imaginal wing discs | Fixed tissue | 10 μM | 30 minutes | Soaking | Alexa Fluor 555 | Genes Dev 26(18):2027-2037 (2012). | |
Maize | Anther | Fixed tissue | 20 μg/mL | 6 hours | N6 culture medium | Alexa Fluor 488 | Plant Physiol 176(2):1610-1626 (2018). |
Xenopus (tadpoles) | Intestine | FFPE tissue | 10 mg/mL | 30–60 min | IP | Alexa Fluor 594 | Cold Spring Harb Protoc 2017(9):pdb.prot097717 (2017). |
Chicken | Cochlea | Fixed whole mount tissue | 50 mg/kg | 4–8 hours | Subcutaneous injection | Alexa Fluor 488 and Alexa Fluor 594 | Laryngoscope 119(9):1770-1775 (2009). |
Nematode (C. elegans) | Germ line cells | Fixed cells | 20 μM | 3–4 hours | Fed through EdU labeled E. coli plates | Click-iT EdU | Genetics 183(1):233-247 (2009). |
A generalized example protocol for EdU detection in FFPE tissue is provided below.
- After incorporation of EdU, isolate the target tissue, fix in formalin, embed in paraffin, section, and mount the tissue on slides using a standard FFPE protocol.
- Deparaffinize the tissue using a standard deparaffinization rehydration protocol. Slides can be placed in a rack and washed in a Coplin jar using the sequential steps below.
Table 2. Deparaffinization and rehydration protocol.
Solution Incubation time Xylene 5 minutes Xylene 5 minutes 100% EtOH 5 minutes 100% EtOH 3 minutes 95% EtOH 3 minutes 85% EtOH 3 minutes 75% EtOH 3 minutes 50% EtOH 3 minutes 1X PBS 5 minutes - Wash tissue in 3% BSA in PBS.
- Detect EdU by the click reaction according to the Click-iT EdU or Click-iT Plus EdU Cell Proliferation Kit. Incubate tissue sections in the Click-iT reaction cocktail for 30 minutes at room temperature, protected from light.
- Wash tissue in 3% BSA in PBS.
- (Optional) Stain the tissue with primary and secondary antibodies for non-EdU protein detection. Wash 3X in 3% BSA in PBS.
- Stain the tissue with Hoechst 33342 (from the Click-iT EdU Cell Proliferation Kit) or other appropriate counterstains. Wash the tissue 3X in PBS.
- Prepare stained tissue in mounting media (such as ProLong antifade mountants) and image slide.
Protocol for dual-labeled EdU and BrdU FFPE tissue
Table 3. Examples of dual BrdU and EdU concentrations and incorporation into animal model systems.
Species | Cell or Tissue type | Cell or Tissue Preparation | EdU/BrdU Conc | EdU/BrdU incubation time | Administration method | Detection | Reference (link) |
---|---|---|---|---|---|---|---|
Mouse | Brain (embryonic) | Frozen tissue | EdU: 20 mg/kg body weight BrdU:200 mg/kg body weight | 1–2 days | IP (pregnant dams) | Alexa Fluor 488 | J Neurosci 31(17):6440-6448 (2011). |
Brain | Fixed tissue | EdU: 7.5 mg/ml, 0.1 ml/10 g mouse BrdU: 7.5 mg/ml, 0.1 ml/10 g mouse` | EdU 0–20 hours then BrdU at 24–44 hours | IP | Alexa Fluor 488 | Mol Biol Cell 22(12):1960-1970 (2011). | |
Skin tumors | Cryosections (fixed) tissue | 6 × 50 μg (both) | EdU: 4 weeks BrdU: 2 hours | IP | Click-iT EdU | Proc Natl Acad Sci U S A 109(52):21468-21473 (2012). | |
Zebra finch | Brain, liver, intestine | Cryosections (fixed) tissue | 50 mg/kg BrdU and 41 mg/kg EdU | 2–8 hours | IM | Alexa Fluor 488 | Biology (Basel) 9(11):356 (2020). |
A generalized protocol for dual pulse EdU-BrdU labeling in FFPE tissue is provided below.
- After incorporation of EdU followed by BrdU in vivo, isolate the target tissue, fix in formalin, embed in paraffin, section, and mount the tissue on slides using a standard FFPE protocol.
- Use a standard deparaffinization rehydration protocol to deparaffinize the tissue (above, table 2).
- Perform antigen retrieval for BrdU detection. A common method is pH 6 citrate-based heat-induced epitope retrieval (HIER), but other methods can be used.
- Perform DNA denaturation for BrdU detection. A common method is incubation in 1-2.5 M HCl at room temperature, but other methods such as treatment with nucleases can be used.
- Neutralize the tissue in 0.1 M sodium borate buffer (pH 8.5) for 10 minutes at room temperature.
- Wash the tissue 3X in 3% BSA in PBS.
- Detect EdU by the click reaction according to the Click-iT EdU or Click-iT Plus EdU Cell Proliferation Kit. Incubate tissue sections in the Click-iT reaction cocktail for 30 minutes at room temperature, protected from light.
- Wash the tissue in 3% BSA in PBS.
- Incubate the tissue with an anti-BrdU primary antibody that does not cross react with EdU (e. g., clone MoBU-1).
- Wash the tissue 3X in 3% BSA in PBS.
- Incubate with appropriate secondary antibody.
- Wash the tissue 3X in 3% BSA in PBS.
- Stain the tissue with Hoechst 33342 (from the Click-iT EdU Cell Proliferation Imaging Kit) or other appropriate counterstains. Wash the tissue 3X in PBS.
- Prepare stained tissue in mounting media (such as ProLong antifade mountants) and image slide.
Appropriate filters for Click-iT EdU Imaging Kits
Table 4. Example filters that can be used with Click-iT EdU Imaging Kits.
Hoechst 33342 | Alexa Fluor 488 | Alexa Fluor 555 | Alexa Fluor 594 | Alexa Fluor 647 | |
---|---|---|---|---|---|
Excitation/Emission (in nm) | 350/461 | 495/519 | 555/565 | 590/615 | 650/670 |
Standard filter set | DAPI | FITC | RFP | TRITC | Cy5 |
Click-iT EdU labeling is compatible with most fixation protocols.
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For Research Use Only. Not for use in diagnostic procedures.