# Staining bugs for micro-CT scanning

[EcoEvoDevoLab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)

Updated 20 November 2024

> Image from [microphotonics.com](https://www.microphotonics.com/x-ray-microscopic-inspection-of-insect-flight-muscles/)

This protocol is intended to visualize thoracic muscle in true bugs. It is based on the reccommendations of [Metscher (2009)](http://www.biomedcentral.com/1472-6793/9/11), who demonstrated the procedure on *Sisyra* sp. (Neuroptera).

### Solutions

#### Bouin's Fixative

Order [from Fisher](https://www.fishersci.com/shop/products/bouin-s-fluid-fixative-4/STLFXBOUPT) or prepare in-house
- Prepare 75 ml saturated aqueous solution of picric acid
- Add 25 ml formalin (37% aqueous solution of formaldehyde)
- Add 5 ml glacial acetic acid

#### I2E Stain

- 1% iodine (I~2~) dissolved in 100% ethanol ([FisherSci](https://www.fishersci.com/shop/products/iodine-alcohol-128-oz/50420830#?keyword=iodine%20in%20ethanol))

#### HMDS

- [1,1,1,3,3,3-hexamethyldisilazane](https://www.fishersci.com/shop/products/1-1-1-3-3-3-hexamethyldisilazane-98-thermo-scientific/AC120581000) (≥97.0%, electron microscopy grade)

### Procedure

#### Fixation

- Anaesthetize insects on CO~2~
- Submnerge in Bouin's Fix for 6 to 18 hours
- Optionally, transfer the tissue to 70% ethanol for storage at 4˚C

#### Staining

- Wash samples twice in 100% ethanol
- Stain in I2E for 24 to 48 hours
- Wash twice times in ethanol
- Wash in HMDS
- Soak in HMDS for 6 to 18 hours
- Air dry in the hood for 16 to 48 hours

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[EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)