TITLE: EdU Immunohistochemistry using Click-it reaction 
AUTHORS: Anita Gola

[DESCRIPTION]
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.

[STEPS]
SECTION: Tissue Preparation and Sectioning
1. Inject animals with EdU as outlined in animal protocol license (typically by performing an intra-peritoneal injection with 200 mg/kg per mouse). 
SECTION: Tissue Preparation and Sectioning
1.1. 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.
SECTION: Tissue Preparation and Sectioning
1.2. The following day, place fixed in tissue in 30% Sucrose Solution (allowing the tissue to get dehydrated).
SECTION: Tissue Preparation and Sectioning
1.3. 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). 
SECTION: Tissue Preparation and Sectioning
1.4. 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).
SECTION: Tissue staining with EdU, antibodies, and Mounting
2. Place tissue slides in humidity chamber, and hydrate tissue sample with PBS.
SECTION: Tissue staining with EdU, antibodies, and Mounting
2.1. Block tissue section using Blocking Buffer (composed with 0.3% Triton, Fc-block,  and 1x BD Perm/Wash (+ Fc-block if needed)). 
SECTION: Tissue staining with EdU, antibodies, and Mounting
2.2. 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). 
SECTION: Tissue staining with EdU, antibodies, and Mounting
2.3. Wash the samples 3 x 5min with PBS
SECTION: Tissue staining with EdU, antibodies, and Mounting
2.4. 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. 
SECTION: Tissue staining with EdU, antibodies, and Mounting
2.5. Mount samples with preferred mounting media (recommended is Fluoromount G).