TITLE: A modified method to analyse cell proliferation using EdU labelling in large insect brains
AUTHORS: Amaia Alcalde Anton, Max S Farnworth, Laura Hebberecht, Jill Harrison, Stephen H Montgomery

[DESCRIPTION]
The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. However, studying this variation requires adapting labelling techniques to visualise cell division in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect. 




[STEPS]
SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
1. Prepare a 10 mM stock solution of EdU: Add 2 mL of DMSO to EdU, then mix well (store at ≤20 °C).

SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
2. Prepare a working solution of the Alexa Fluor® azide: Add 70 µL of DMSO to the Alexa Fluor® azide (store at ≤20 °C).

SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
3. Prepare a working solution of 1X Click-iT® EdU reaction buffer: Transfer all of the solution (4 mL) in the 1X Click-iT® EdU reaction buffer bottle to 36 mLof deionized water. Store at4 °C.

SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
4. To create the 10X stock solution of the Click-iT® EdU buffer additive, add 2 mL of deionized water to the vial (store at ≤ 20 °C).

SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
5. Prepare paraformaldehyde, PFA (4% PFA in [0.1 M] phosphate-buffered saline (PBS; 7.4 pH). Alternatively, zinc-formaldehyde, ZnFA (0.25% [18.4 mM] ZnCl2; 0.788% [135 mM] NaCl; 1.2% [35 mM] sucrose; 1% formaldehyde) can also be prepared and used. Store at 4 Room temperature.
SECTION: 0.	BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
6. Prepare the permeabilization buffer (1 % Triton ® X-100 in [0.1 M] PBS). Store at 4 °C.
SECTION: 1.	EDU INCORPORATION
7. Prepare the EdU working solution. Dilute EdU in Grace’s Medium (GM, ThermoFisher, #11595030) to a final concentration of 20µM: add 20 µL from the stock solution (prepared in step 0.1) to 10 mL of GM. Make 1 mL aliquots (store at ≤ 20 °C).

SECTION: 1.	EDU INCORPORATION
8. A)    Ex vivo brain incubation 


    

SECTION: 1.	EDU INCORPORATION
8.1. Dissect out the brain in 50% Grace’s medium diluted in [0.1 M] phosphate-buffered saline (PBS; pH
7.4). 

 

SECTION: 1.	EDU INCORPORATION
8.2. Incubate the brain in a solution of EdU diluted in Grace’s medium for 1-2h. 

 

SECTION: 1.	EDU INCORPORATION
9. B)    In vivo incubation 


    

SECTION: 1.	EDU INCORPORATION
9.1. For active life stages, chill individuals in a refrigerator for a couple of minutes to anesthetize them. When ready, open a small window in the cuticle. 



SECTION: 1.	EDU INCORPORATION
9.2. Incubate the brain in a solution of EdU diluted in Grace’s medium for 1-2h.

SECTION: 1.	EDU INCORPORATION
9.3. After the incubation, finish the dissection in HEPES-buffered saline, HBS ([150 mM] NaCl;
[5 mM] KCl; [5 mM] CaCl2; [25 mM] sucrose; [10 mM HEPES]; pH 7.4).
SECTION: 2.	FIXATION
10. Transfer the brains to a 12 well plate, with up to 5 brains per well. 
SECTION: 2.	FIXATION
11. Fix the brain in a solution of paraformaldehyde, PFA (4% PFA in [0.1 M] phosphate-buffered saline (PBS; 7.4 pH) for 6-14h (Depending on the stage 14h for adults, 6-10h for larval stages). 

 


SECTION: 2.	FIXATION
12. Remove the fixative and wash each well three times for 10 min with 1 mL of 0.1% Triton® X-100 in [0.1 M] PBS, 0.1%PBS-T.

 


SECTION: 3. PERMEABILIZATION
13. Remove the PBS solution (from step 12).
SECTION: 3. PERMEABILIZATION
14. Add 1 mL of 1% Triton® X-100 in [0.1 M] PBS, 1%PBS-T to each well. 

SECTION: 3. PERMEABILIZATION
15. Incubate at room temperature for 2h for whole brains, or 30 minutes for
sectioned tissue. 

SECTION: 4. CLICK IT REACTION
16. Dilute 1X Click-iT® EdU buffer additive in deionized water at a ratio of 1:10. This should be prepared fresh and used on the same day.
SECTION: 4. CLICK IT REACTION
17. Remove the permeabilization buffer (from step 3.15).
SECTION: 4. CLICK IT REACTION
18. Wash each well three times with 0.1% Triton® X-100 in [0.1 M] PBS, 0.1%PBS-T.
SECTION: 4. CLICK IT REACTION
19. Remove the wash solution.
SECTION: 4. CLICK IT REACTION
20. Prepare the Click-IT® reaction mix. Add the ingredients in the order defined by the manufacturer, replicated below. Use the Click-iT® reaction mix within 15 minutes of preparation.

Table 1. Click-iT® reaction cocktail. All the components should be added in the order listed below. Table from Click-iT® EdU Imaging Kits Protocol, ThermoFisher.


   

SECTION: 4. CLICK IT REACTION
21. Add 0.5 mL of the Click-iT® reaction cocktail to each well containing up to 3 brains, and 1 mLto each well containing 4-5 brains. Make sure the brains are completely covered in the reaction mix.

SECTION: 4. CLICK IT REACTION
22. Incubate the plate for 30 minutes at room temperature, protect from light using aluminium foil.

SECTION: 4. CLICK IT REACTION
23. Remove the reaction mix.
SECTION: 4. CLICK IT REACTION
24. Wash each well three times with 1 mL of 0.1% PBS-T (10 minutes each).

SECTION: 4. CLICK IT REACTION
25. Remove the wash solution.

 

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27. B) DAPI
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.8. Wash each well three times with 1 mL of 0.1% PBS-T.

SECTION: 6. TISSUE CLARIFICATION
28. 
 


Incubate the brains under agitation in progressively more concentrated glycerol solutions: 1%, 2%, 4% (2 h each), 8%, 15%, 30%, 50%, 60%, 70%, and 80% (1 hour each) glycerol diluted in 0.1M Tris buffer, with DMSO to 1% final concentration.

SECTION: 6. TISSUE CLARIFICATION
29. Wash the brains with 100% ethanol for 30 minutes under agitation, repeat three times.

SECTION: 6. TISSUE CLARIFICATION
30. Transfer each brain in a small amount of ethanol (200-400 µL) to an Eppendorf tube.

SECTION: 6. TISSUE CLARIFICATION
31. Underlay the ethanol with methyl salicylate, wait for brain to sink with no agitation.
SECTION: 6. TISSUE CLARIFICATION
32. Aspirate the fluid from the top down, replace with fresh methyl salicylate and allow the brain to sink again for ~30 minutes with no agitation.

SECTION: 6. TISSUE CLARIFICATION
33. Aspirate the fluid and mount in fresh methyl salicylate.
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.7. Wash each well with 1 mL of miliQ H2O with 0.2% Triton for 10 minutes.

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.6. Remove the DAPI solution.
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.5. Add 1 mL of diluted DAPI solution per well. Incubate for 2-3 hours for whole brains, or 30 minutes for sectioned tissue, at room temperature, protected from light. 



SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.4. Dilute the DAPI solution in miliQ H2O at a ratio of 1:1000.
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.3. Wash once in milliQ/deionized H2O with 0.2% Triton, 10 minutes.

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.2. Remove the wash solution
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
27.1. Wash each well twice with 1 mL of [0.1 M] PBS (30 minutes each).

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.6. Remove the wash solution.
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.5. Wash each well twice with 1 mL of PBS.

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.4. Add 1 mL of 1X Hoechst 33342 solution per well. Incubate for 2-3 hours for whole brains, or 30 minutes for sectioned tissue, at room temperature, protected from light. 

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.3. Dilute the Hoechst 33342 solution in [0.1 M] PBS at a ratio of 1:2000 to obtain a 1X Hoechst 33342 solution (the final concentration is 5 µg/mL).
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.2. Remove the wash solution.
SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26.1. Wash each well twice with 1 mL of [0.1 M] PBS (30 minutes each).

SECTION: NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
26. A) Hoechst 33342