Mar 20, 2023

EdU Immunohistochemistry using Click-it reaction 

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbyoy1vpk/v1
  • 1Rockefeller University
Anita Gola
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbyoy1vpk/v1
Protocol Citation: Anita Gola 2023. EdU Immunohistochemistry using Click-it reaction . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbyoy1vpk/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 18, 2023
Last Modified: March 20, 2023
Protocol  Integer ID: 79033
Keywords: phase cells with the thymidine analogue, thymidine analogue, modified thymidine analogue, specific click reaction, using basic immunohistochemistry technique, antibody method, using click, basic immunohistochemistry techniques in combination, compatible with antibody method, immunohistochemistry, photostable alexa fluor, antibody, histology tissue, fluorescent labeling, click, assay, edu capture, proliferating cell, synthesized dna
Abstract
This protocol allows users to image histology tissue using basic immunohistochemistry techniques in combination with EdU capture (using the Click-it reaction system). EdU (5-ethynyl-2′-deoxyuridine) will capture S-phase cells with the thymidine analogue, allowing to take a snap-shot of actively diving cells. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor™ dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.
Materials
  1. dU 5-ethynyl-2′-deoxyuridine (200mg/kg/mouse)
  2. BD Fix/Perm Solution (cat #: 554714)
  3. 30% Sucrose Solution
  4. Tissue-Tek O.C.T. Compound (cat#: 4583; Sakura)
  5. Tissue-Tek Base Mold System (Sakura): size will depend on organ of interest
  6. SuperFrost Plus slides (cat #: 12-550-15; Fisher)
  7. Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 555 dye (cat #: C10338; Thermo): A488, A555, A594, A647 work very well!
  8. Optional: TruStain FcX™ (anti-mouse CD16/32) Antibody (cat#: 101319; Biolegend)
  9. Optional: primary/secondary antibody needed for wanted panel
  10. Optional DAPI
  11. Optional Flouromount G (cat #: 00-4958-02, Thermo)

Tissue Preparation and Sectioning
1d 4h 30m
Inject animals with EdU as outlined in animal protocol license (typically by performing an intra-peritoneal injection with 200 mg/kg per mouse).
30m
Euthanize animal and harvest organ of interest: place tissue in 0.1% BD cytofix/cytoperm fixative (0/N at 4C).
Time of euthanasia after EdU injection will vary widely on organ/cell of interest, and age of the animal. For an optimal time window- prior literature should be investigated. For fast proliferating tissues (such as intestinal epithelium or skin), a good starting point will be around 1-4hrs; for developing embryos, 20-30min might be sufficient.
4h
The following day, place fixed in tissue in 30% Sucrose Solution (allowing the tissue to get dehydrated).
1d
Embed tissue in Tissue-Tek Base Mold System by orienting the tissue in its optimal configuration in OCT Mounting Media. Freeze tissue on dry ice until OCT turns from a viscous clear compound to a solid white. OCT blocks can be stored at -80C long term (months).
1h
Section OCT blocks on a cryostat at desired thickness on SuperFrost Plus slides. Tested so far are up to 50um in thickness. If desired, sectioned slides can be stored at -20C long term (months).
2h
Tissue staining with EdU, antibodies, and Mounting
1h 5m
Place tissue slides in humidity chamber, and hydrate tissue sample with PBS.
5m
Block tissue section using Blocking Buffer (composed with 0.3% Triton, Fc-block, and 1x BD Perm/Wash (+ Fc-block if needed)).
1h
Stain sample with EdU Click-it reaction solution following the instructions from the manufacturer. EdU staining is extremely bright- 30min will be sufficient to stain efficiently a 25um section. 45min staining is advised for thicker sections. The copper concentrations typically used in traditional click chemistry reactions can affect fluorophores such as GFP, mCherry, R-PE, and R-PE tandem dyes. Highly recommend to 1) stain for EdU before other antibodies (either conjugated-primaries or secondaries), and 2) stain with anti-fluorophore antibodies (such as anti-GFP).
30m
Wash the samples 3 x 5min with PBS
15m
Continue staining samples with wanted panel (primary, secondary and/or Nuclear Dye (such as DAPI)). For follow-up quantification (for example: % EdU+ cells), DAPI staining is very much important.
1d
Mount samples with preferred mounting media (recommended is Fluoromount G).
30m