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Table 3 Notes on fixatives

From: MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues

Fixative

Notes

neutral-buffered formalin (10% NBF)

Formalin = 37% formaldehyde solution (aq.).

 

Normally used at 10% dilution in phosphate buffer at pH 7.0

 

Commercial formalin usually contains about 10% methanol.

 

The most common, but rarely the best fixative. [23, 24]

paraformaldehyde

Polymerized formaldehyde, usually dissolved in buffer (e.g. PBS) at 4% w/v when a chemically-controlled fixative is required.

 

Action is generally similar to 10% NBF. [23, 24]

gluteraldehyde

Strong cross-linking fixative, often prepared in cacodylate buffer or a less toxic alternative such as HEPES. Common fixative for electron microscopy. [23, 24]

4F1G

4% (or 3.7%) formaldehyde + 1% gluteraldehyde in phosphate buffer.

 

Takes advantage of the faster penetration of formaldehyde and the superior fixing action of gluteraldehyde. Common fixation for electron microscopy. [25]

Bouin's fluid

75 parts (v/v) saturated aqueous picric acid,

 

25 parts formalin (37% formaldehyde),

 

5 parts glacial acetic acid.

 

A standard and excellent histological fixative. [24]

alcoholic Bouin's

Refers to either a mixture of Bouin's fluid and ethanol (1:1), or to the fixative also known as Bouin-Duboscq-Brasil [24]. The two are similar in final composition.

 

The alcoholic solutions penetrate more readily and are sometimes favored for arthropods.

glyoxal

A cross-linking dialdehyde (OCHCHO) prepared in acidic buffers and marketed as formalin substitutes: Prefer (Anatech Ltd.; http://www.anatechltdusa.com) and Shandon Glyo-Fixx (Thermo Scientific; http://www.thermo.com).

 

Much less volatile and toxic than formaldehyde.

 

Very good tissue preservation; especially good for immunostaining.

Dent's fixative

80% methanol, 20% DMSO

 

Rapid dehydrating fixative. Expect some tissue shrinkage. Often used for immunostaining.

hot alcohol

Samples are dropped into 70% ethanol at about 60°C.

 

Mainly used for fixing soft-bodied animals, such as insect larvae and pupae.

  1. In general, the best fixative for microCT scanning will be the best histological fixative for the particular tissues under investigation.