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:

You will need the following for this protocol:

Reagents

Protocol

EdU labeling in FFPE tissue

Table 1. Examples of EdU concentrations and incorporation into animal model systems.

SpeciesCell or Tissue typeCell or Tissue PreparationEdU ConcEdU incubation timeAdministration methodDetectionReference (link)
MouseColonFrozen, fixed tissue1 mg/mouse4–144 hoursIPAlexa Fluor 488J Immunol 188(5):2427-2436 (2012).
Small intestine and brainFFPE (brain and small intestine), fresh, fixed (small intestine) tissue100–200 μg24–96 hoursIPAlexa Fluor 568 and TMRProc Natl Acad Sci U S A 105(7):2415-2420 (2008).
Retina (dissociated cells)Fixed cells25 μg/g4 hoursIPAlexa Fluor 647Mol Biol Cell 23(22):4362-72 (2012).
Embryonic brainCryosection tissue5 µg/g24–72 hoursIP (pregnant dams)Alexa Fluor 594Proc Natl Acad Sci U S A 19;110(8):3113-8 (2013).
organ of Corti (cochlear neurosensory epithelia)Whole mount tissue50 mg/kg4 hoursSubcutaneous injectionAlexa Fluor 488J Neurosci 31(24):8883-93 (2011).
Optic nervesCryosection, fixed tissue0.2 mg/mL2–8 weeks (water exchanged every 72 hours)Drinking waterAlexa Fluor 594J Neurosci 32(36):12528-42 (2012).
RatLungCryosection, fixed tissue50 mg/kg24 hoursIPAlexa Fluor 488Anal Cell Pathol (Amst) 2015:326385 (2015).
Olfactory EpitheliumCryosection, fixed tissue50 mg/kg2 hoursSubcutaneousAlexa Fluor 594J Neurosci 38(21):5022-5037 (2018).
Bone (HSPCs from bone marrow)Fresh and fixed cells60 mg/kg24 hoursIP (pregnant dams)Alexa Fluor 647, Alexa Fluor 488 for flowNat Commun 13(1):1327 (2022).
ZebrafishLarval intestineFFPE tissue100 μg/mL16 hoursSoakingAlexa Fluor 488Proc Natl Acad Sci U S A 108 Suppl 1(Suppl 1):4570-4577 (2011).
Larval jawsFixed tissue400 μM24 hoursSoakingClick-iT EdUFASEB J 35(11):e22002 (2021).
Cardiac tissueCryosection, fixed tissue10 mM3 daysIPClick-iT EdUElife 10:e66079 (2021).
DrosophilaLarval brain, eye discs & tracheaFixed tissue20–100 μM10–60 minutesSoakingAlexa Fluor 594Development 138(23):5201-5212 (2011).
Larval imaginal wing discsFixed tissue10 μM30 minutesSoakingAlexa Fluor 555Genes Dev 26(18):2027-2037 (2012).
MaizeAntherFixed tissue20 μg/mL6 hoursN6 culture mediumAlexa Fluor 488Plant Physiol 176(2):1610-1626 (2018).
Xenopus (tadpoles)IntestineFFPE tissue10 mg/mL30–60 minIPAlexa Fluor 594Cold Spring Harb Protoc 2017(9):pdb.prot097717 (2017).
ChickenCochleaFixed whole mount tissue50 mg/kg4–8 hoursSubcutaneous injectionAlexa Fluor 488 and Alexa Fluor 594Laryngoscope 119(9):1770-1775 (2009).
Nematode (C. elegans)Germ line cellsFixed cells20 μM3–4 hoursFed through EdU labeled E. coli platesClick-iT EdUGenetics 183(1):233-247 (2009).

A generalized example protocol for EdU detection in FFPE tissue is provided below.

  1. 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.
  2. 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.

    SolutionIncubation time
    Xylene5 minutes
    Xylene5 minutes
    100% EtOH5 minutes
    100% EtOH3 minutes
    95% EtOH3 minutes
    85% EtOH3 minutes
    75% EtOH3 minutes
    50% EtOH3 minutes
    1X PBS5 minutes

  3. Wash tissue in 3% BSA in PBS.
  4. 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.
  5. Wash tissue in 3% BSA in PBS.
  6. (Optional) Stain the tissue with primary and secondary antibodies for non-EdU protein detection. Wash 3X in 3% BSA in PBS.
  7. Stain the tissue with Hoechst 33342 (from the Click-iT EdU Cell Proliferation Kit) or other appropriate counterstains. Wash the tissue 3X in PBS.
  8. 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.

SpeciesCell or Tissue typeCell or Tissue PreparationEdU/BrdU ConcEdU/BrdU incubation timeAdministration methodDetectionReference (link)
MouseBrain (embryonic)Frozen tissue

EdU: 20 mg/kg body weight

BrdU:200 mg/kg body weight

1–2 daysIP (pregnant dams)Alexa Fluor 488J Neurosci 31(17):6440-6448 (2011).
BrainFixed 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 hoursIPAlexa Fluor 488Mol Biol Cell 22(12):1960-1970 (2011).
Skin tumorsCryosections (fixed) tissue6 × 50 μg (both)

EdU: 4 weeks

BrdU: 2 hours

IPClick-iT EdUProc Natl Acad Sci U S A 109(52):21468-21473 (2012).
Zebra finchBrain, liver, intestineCryosections (fixed) tissue50 mg/kg BrdU and 41 mg/kg EdU2–8 hoursIMAlexa Fluor 488Biology (Basel) 9(11):356 (2020).

A generalized protocol for dual pulse EdU-BrdU labeling in FFPE tissue is provided below.

  1. 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.
  2. Use a standard deparaffinization rehydration protocol to deparaffinize the tissue (above, table 2).
  3. 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.
  4. 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.
  5. Neutralize the tissue in 0.1 M sodium borate buffer (pH 8.5) for 10 minutes at room temperature.
  6. Wash the tissue 3X in 3% BSA in PBS.
  7. 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.
  8. Wash the tissue in 3% BSA in PBS.
  9. Incubate the tissue with an anti-BrdU primary antibody that does not cross react with EdU (e. g., clone MoBU-1).
  10. Wash the tissue 3X in 3% BSA in PBS.
  11. Incubate with appropriate secondary antibody.
  12. Wash the tissue 3X in 3% BSA in PBS.
  13. 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.
  14. 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 33342Alexa Fluor 488Alexa Fluor 555Alexa Fluor 594Alexa Fluor 647
Excitation/Emission (in nm)350/461495/519555/565590/615650/670
Standard filter setDAPIFITCRFPTRITCCy5

Click-iT EdU labeling is compatible with most fixation protocols.

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For Research Use Only. Not for use in diagnostic procedures.