Jul 10, 2023

Public workspaceA modified method to analyse cell proliferation using EdU labelling in large insect brains

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.n92ldmy69l5b/v1
  • 1School of Biological Sciences, University of Bristol, BS8 1TQ, UK
  • PLOS ONE Lab Protocols
    Tech. support email: plosone@plos.org
Amaia Alcalde Anton
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DOI: dx.doi.org/10.17504/protocols.io.n92ldmy69l5b/v1
External link: https://doi.org/10.1371/journal.pone.0292009
Protocol Citation: Amaia Alcalde Anton, Max S Farnworth, Laura Hebberecht, Jill Harrison, Stephen H Montgomery 2023. A modified method to analyse cell proliferation using EdU labelling in large insect brains. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmy69l5b/v1
Manuscript citation:
Anton AA, Farnworth MS, Hebberecht L, Harrison CJ, Montgomery SH (2023) A modified method to analyse cell proliferation using EdU labelling in large insect brains. PLOS ONE 18(10): e0292009. https://doi.org/10.1371/journal.pone.0292009
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: July 04, 2023
Last Modified: July 10, 2023
Protocol Integer ID: 84483
Keywords: cell proliferation, neurogenesis, mitosis, neurodevelopment
Funders Acknowledgement:
ERC Starter Grant
Grant ID: (758508)
NERC IRF
Grant ID: (NE/N014936/1)
Disclaimer
This protocol has been modified from the instructions of the Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye - Thermofisher. Note that some steps (e.g. prepare the stock solutions) have not been changed.
Abstract
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.
Image Attribution
Amaia Alcalde Antón
Safety warnings
EdU, 5-ethynyl-2'-deoxyuridine
  • Hazard: Irritant
  • Exposure Route(s): inhalation, ingestion, injection, absorption
  • First Aid: EYE CONTACT May cause eye irritation with susceptible persons. SKIN CONTACT May cause skin irritation in susceptible persons. INHALATION May be harmful by inhalation. INGESTION May be harmful if swallowed.

DMSO Dimethyl sulfoxide
  • Hazard: Irritant
  • Exposure Route(s): Spills, splashing
  • First Aid: SKIN CONTACT Rinse skin with water. Immediate medical attention is not required. EYE CONTACT Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

Hoechst 33342
  • Hazard: Mutagen
  • Exposure Route(s): Spills, splashing
  • First Aid: SKIN CONTACT Rinse skin with water. Immediate medical attention is not required. EYE CONTACT Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

DAPI - 4',6-Diamidino-2-phenylindole dihydrochloride
  • Hazard: Irritant
  • Exposure Route(s): Spills, splashing
  • First Aid: SKIN CONTACT Rinse skin with water. Immediate medical attention is not required. EYE CONTACT Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

0. BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
0. BEFORE YOU START – PREPARE THE STOCK SOLUTIONS
Prepare a 10 mM stock solution of EdU: Add Amount2 mL of DMSO to EdU, then mix well (store at ≤Temperature20 °C ).

Prepare a working solution of the Alexa Fluor® azide: Add Amount70 µL of DMSO to the Alexa Fluor® azide (store at ≤Temperature20 °C ).

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 Amount36 mL of deionized water. Store atTemperature4 °C .

To create the 10X stock solution of the Click-iT® EdU buffer additive, add Amount2 mL of deionized water to the vial (store at ≤ Temperature20 °C ).

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 TemperatureRoom temperature .
Prepare the permeabilization buffer (1 % Triton ® X-100 in [0.1 M] PBS). Store at Temperature4 °C .
1. EDU INCORPORATION
1. EDU INCORPORATION
Prepare the EdU working solution. Dilute EdU in Grace’s Medium (GM, ThermoFisher, #11595030) to a final concentration of 20µM: add Amount20 µL from the stock solution (prepared in step 0.1) to Amount10 mL of GM. Make 1 mL aliquots (store at ≤ Temperature20 °C ).

A) Ex vivo brain incubation




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

Note
This should be done as quickly as possible.

Incubate the brain in a solution of EdU diluted in Grace’s medium for 1-2h.

Note
The time of incubation will affect the number of cell cycles which are exposed to EdU, and therefore longer incubations will likely lead to more labelled cells in mitotically active tissue.

B) In vivo incubation




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.
Incubate the brain in a solution of EdU diluted in Grace’s medium for 1-2h.

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).
2. FIXATION
2. FIXATION
Transfer the brains to a 12 well plate, with up to 5 brains per well.
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).
Note
Alternatively, zinc-formaldehyde, ZnFA (0.25% [18.4 mM] ZnCl2; 0.788% [135 mM] NaCl; 1.2% [35 mM] sucrose; 1% formaldehyde) can be used to fix the brains 14-16h (14h for larval brains, 16h for adult brains).


Remove the fixative and wash each well three times for 10 min with Amount1 mL of 0.1% Triton® X-100 in [0.1 M] PBS, 0.1%PBS-T.

Note
If tissue sectioning is required, we suggest performing it at this stage.


3. PERMEABILIZATION
3. PERMEABILIZATION
Remove the PBS solution (from step 12).
Add Amount1 mL of 1% Triton® X-100 in [0.1 M] PBS, 1%PBS-T to each well.

Incubate at room temperature for 2h for whole brains, or 30 minutes for sectioned tissue.

4. CLICK IT REACTION
4. CLICK IT REACTION
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.
Remove the permeabilization buffer (from step 3.15).
Wash each well three times with 0.1% Triton® X-100 in [0.1 M] PBS, 0.1%PBS-T.
Remove the wash solution.
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.




Add Amount0.5 mL of the Click-iT® reaction cocktail to each well containing up to 3 brains, and Amount1 mL to each well containing 4-5 brains. Make sure the brains are completely covered in the reaction mix.

Incubate the plate for 30 minutes at room temperature, protect from light using aluminium foil.

Remove the reaction mix.
Wash each well three times with Amount1 mL of 0.1% PBS-T (10 minutes each).

Remove the wash solution.

Note
Additional immunohistochemical staining can be performed at this point.

NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
NUCLEAR STAINING with Hoechst 33342 or DAPI Staining
A) Hoechst 33342

Wash each well twice with Amount1 mL of [0.1 M] PBS (30 minutes each).

Remove the wash solution.
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).
Add Amount1 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.

Wash each well twice with Amount1 mL of PBS.

Remove the wash solution.
B) DAPI
Wash each well twice with 1 mL of [0.1 M] PBS (30 minutes each).

Remove the wash solution
Wash once in milliQ/deionized H2O with 0.2% Triton, 10 minutes.

Dilute the DAPI solution in miliQ H2O at a ratio of 1:1000.
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.



Remove the DAPI solution.
Wash each well with 1 mL of miliQ H2O with 0.2% Triton for 10 minutes.

Wash each well three times with Amount1 mL of 0.1% PBS-T.

6. TISSUE CLARIFICATION
6. TISSUE CLARIFICATION
2h
2h

Note
Clarification methods can vary. In the current study we followed Ott (2008).


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.

Wash the brains with 100% ethanol for 30 minutes under agitation, repeat three times.

Transfer each brain in a small amount of ethanol (Amount200-400 µL ) to an Eppendorf tube.

Underlay the ethanol with methyl salicylate, wait for brain to sink with no agitation.
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.

Aspirate the fluid and mount in fresh methyl salicylate.
Protocol references
Clarification section:

Ott SR. Confocal microscopy in large insect brains: Zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts. J Neurosci Methods. 2008;172(2):220–30.