Methods to study organogenesis in decapod crustacean larvae. I. larval rearing, preparation, and fixation

Table 3 Recommended fixatives discriminated by suitability for each specific method

Histological technique	Recommended fixatives	Acceptable fixatives
External observation: light microscopy and epifluorescence	PFA, FASW, Karnovsky’s	75% ethanol
Immunohistochemistry	PFA	FASW
Micro CT	Bouin’s, PFA	FASW, Karnovsky’s, 75% ethanol
Scanning electron microscopy	FAE, AAF	75% ethanol
Paraffin histology	Bouin’s, Karnovsky’s	FAE, PFA
Semithin sectioning	Bouin’s, Karnovsky’s or FAE followed by OS
Transmission electron microscopy	Karnovsky’s, GA or TA, followed by OS; GAOS

AAF: 85 ml 100% ethanol, 5 ml glacial acetic acid, 10 ml 37% formaldehyde*
Bouin’s fixative: 10% formaldehyde, 5% glacial acetic acid in saturated aqueous picric acid 1.2%*
FAE: 150 ml 80% ethanol, 60 ml 37% formalin, and 15 ml glacial acetic acid*
FASW: 4% formaldehyde in water from the animal’s habitat
GA: 4% glutardialdehyde in 0.1 M phosphate or cacodylate-buffered saline pH 7.1–7.3*
GAOS: 2–4% glutardialdehyde, 1–2% OsO₄ in 0.1 M cacodylate buffer pH 7.1–7.2*
Karnovsky’s solution: 2–4% formaldehyde and glutaraldehyde in 0.1 M buffer (phosphate buffer or cacodylate buffer) pH 7.1 to 7.2*
OS: 0.5–4% osmium tetroxide (OsO₄) in 0.1 M buffer*
PFA: 4% paraformaldehyde in 0.1 M phosphate-buffered saline pH 7.4*
TA: 2% tannin, 2% glutardialdehyde in 0.1 M sodium cacodylate buffer pH 7.2*
*Adjust osmolarity as described in "Preparation and fixation" section

ISSN: 1438-3888