Mar 03, 2026
EdU Immunohistochemistry: Chromogen detection of thaw-mounted sections
- Tracy Larson1
- 1Department of Biology, University of Virginia
Protocol Citation: Tracy Larson 2026. EdU Immunohistochemistry: Chromogen detection of thaw-mounted sections. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g77nb1gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 02, 2026
Last Modified: March 03, 2026
Protocol Integer ID: 244279
Keywords: neural precursor cells, radial glia, neurogenesis, immunohistochemistry, chromogen stain, songbird, edu cell proliferation kit, it kit for edu cell proliferation kit, edu immunohistochemistry, need for dna denaturation, thymidine analog, chromogen detection, chromogen detection of thaw, edu detection, proliferating cell, brdu antigen retrieval, dna denaturation, synthesized dna, invitrogen click, state antibody, dna, mitosis, antigen
Funders Acknowledgements:
NIGMS
Grant ID: 1R35GM150562
Abstract
EdU (5-ethynyl-2′-deoxyuridine) is a high-speed, reportedly-safe method for detecting proliferating cells by incorporating a thymidine analog into newly synthesized DNA during S phase of mitosis. EdU detection utilizes "click chemistry" for detection, eliminating the need for DNA denaturation and better preserving cellular morphology and antigens when compared to BrdU antigen retrieval and labeling. This protocol utilizes the Invitrogen Click-IT kit for EdU Cell Proliferation Kit and can be multiplexed with additional lineage or state antibodies as desired.
Materials
Solutions and reagents:
10xPBS, phosphate buffered saline
To make 1 L of 10x stock:
NaCl 80.0 g
KCl 2.0 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
distilled water to 1.0 L
pH to 7.4
*Dilute to 1x working concentration with distilled water
5% BSA Immunoblock
BSA 5 mg
1x PBS to 100 mL
*Store at -20C long-term. Keep at 4C for up to a week of use.
Endogenous Peroxidase Solution
30% H2O2 50 ul
PBS to 50 ml
*Make fresh just before use
1x Click-It EdU Reaction Buffer (Invitrogen, C10269)
Dilute from 10x to 1x with dH2O
*This can be substituted with 1xTBS
Reaction Buffer Additive (Invitrogen, C10644 [kit])
Add 2 mL of dH2O to vial and store at -20C; stable up to 1 yr
Biotin Azide Stock (Invitrogen, B10184)
Add 70 μl DMSO, mix well, and store at -20C; stable up to 1 yr
*Note: if a previous Alexo Fluor kit has been purchased, the biotin azide can substitute for the Alexa Fluor azide
DAB with Nickle (Black) Stain
DAB (40 mg/mL) 300 ul
30% H2O2 30 ul
0.5% nickle solution 3 ml
1x TBS to 30 ml
*Make fresh just before use and pH should be around 7.5
EdU Developing solution
1x Click it Reaction buffer or 1x TBS 430 μl
Cuso4(H) 20 μl
Biotin Azide (for final 1:2000) 0.25 μl
10x Rxn buffer Additive 50 μl
---------
500 μl
*Scale for number of slides to incubate (200 - 250ul each slide). Use within 15 minutes of preparation
0.5% Nickel Solution
nickel sulfate 0.5 g
distilled water 100 mL
*pH to around 8
10x TBS
Tris-base 30 g
NaCl 80 g
KCl 2 g
dH2O to 1L
*pH to 7.6, dilute to 1x working concentration
Troubleshooting
Safety warnings
DAB is a carcinogen and should be handled with appropriate PPE and waste handling procedures.
Before start
For positive control of EdU incorporation and staining, collect and section gut tissue from an animal of the same species pulsed with a 2 hr pre-pulse of EdU. Cross sections of tissue will contain rings of dividing cells that can visualized by eye during chromogenic staining.
Antigen Retrieval and Blocking Endogenous Activity
Fix thaw mounted, 40um thick sections for 15 minutes at room temperature in 4% paraformaldehyde
Remove fixative and incubate slides with 15% MEOH in 1x PBS for 30 minutes at -20C
Incubate in peroxidase solution to remove endogenous peroxidase activity from tissue. Be sure to mix solution well and incubate tissue in solution for 30 minutes. Periodically reduce bubbles formation by tapping coplin jars on counter.
Rinse in 1x PBS, two times for 10 min
Prepare incubation chamber: using a flat bottom food storage container (with lid), place a pre-fit eggcrate lighting louver over paper towels into chamber. Pre-wet with PBS.
Block endogenous avidin and biotin activity with the Avidin Biotin kit (Vector). Dilute 4 drops per 1mL in PBS, adding 400ul to each slides laid flat on top of louver in chamber. Incubate for 30-45min, sequentially with a rinse of PBS rinse in between the incubations.
Block with 3-5% BSA in PBS for 1 hr at room temperature
Change block with EdU developing solution. Incubate 2-3 hrs at room temperature or overnight at 4C
Wash four times with 1xPBS, 10 minutes each
Incubate for 45 minutes at room temperature, with ABC-elite mix. As per the manufacturer's instructions, mix solution 30 min in advance to “activate” by adding 1 drop of A, 1 door of B per 5mL of 1x PBS. Incubate with slides in incubation chamber, with 400uL pipetted onto each slide.
Remove slides from the incubation chamber and rinse one time in 1x PBS for 15 minutes
Rinse one time in 1% EDTA solution (dissolved in water, pH 8.0-8.5) for ten minutes. This step reduces weakly-bound copper from the EdU solution that remains in tissue and will cause subsequent background staining.
Rinse three times in 1X PBS for 15 minutes each
Mix DAB staining solution with nickel and immediately add 800-1000uL to each slide. Watch for desired staining using the positive control of one slide of BrdU-labeled gut tissue. Once preferred staining is achieved, stop the reaction by dipping in H2O.
Note: Use a dedicated incubation chamber with louver to prevent contamination with DAB (a carcinogen). Use appropriate PPE and waste handling procedures.
Note: Remaining copper from the EdU staining solution will produce a green-ish, grey color in the absence of nickel in addition to background staining. Background can be removed subsequently.
(optional) Following DAB staining, if background persists rinse for 10 minutes or longer (to remove any additional background) in 1% EDTA solution (dissolved in water, pH 8.0-8.5). Note: this will begin to reduce the darkness of all stain, so use cautiously to enhance the signal to noise ratio.
Rinse slides in 1x PBS three times for 10 minutes each
Antibody #2
(optional) A second antigen can be used as desired.
Mounting Slides
In a fume hood:
75% ethanol for 3 minutes
95% ethanol for 3 minutes
100% ethanol for 3 minutes
Xylene for 2 minutes
Apply a sufficient amount of DPX Mountant to each slide and gently lay a No.1 22x60mm glass coverslip on top of DPX. Gently adjust coverslip to ensure that it is not hanging over any edges. Note: place slides prior to DPX onto cardboard covered with two paper towels. Tape the edges of the towels so they are taught. These paper towels prevent slides from sticking to surface.
Leave in fume hood at room temperature for slides to dry, keeping flat. If paper towel or extra DPX remains on glass after dry, it can be removed by scraping off with a razor blade.