For over a century, the gold standard for fixation of histological samples is characterized by a solution of formaldehyde in water, also known as 10% formalin. Although this fixative has notable merits in preserving the morphology of cells and tissues, environmental authorities are increasingly concerned about the objective toxicity of formalin. As a volatile reagent, formalin displays allergenic, neurotoxic, and cancerogenic properties. In recent years, alternative fixatives have been considered as substitutes to formalin with the aim to reduce the described negative effects. In particular, fixatives utilizing a glyoxal solution deprived of acids, have garnered attention due to their non-volatile nature and lack of carcinogenic hazard. Although glyoxal acid-free (GAF®) has been tested on tissues from human and terrestrial animals, there is a lack of knowledge regarding its efficacy in fish species.
The aim of this study was to test the performance of GAF® fixative in comparison with those of two distinct fixatives: 10% formalin and 4% paraformaldehyde. Two different formulations of glyoxal acid-free (GAF®) have been considered: the first, previously tested on samples from terrestrial animals, while the second was specifically formulated for samples from fish species. For the trial, we selected zebrafish (Danio rerio) to assess fixation in freshwater fishes, and European sea bass (Dicentrarchus labrax) as marine species. Various target organs such as liver, muscle, gills, and gonads were sampled in adults of both species; moreover, in zebrafish, whole animals as well as heads and trunks were collected. The samples were maintained in the respective fixatives for 48 hours. The preservation of morphology was evaluated on histological sections by using haematoxylin-eosin as well as Masson’s trichrome staining. Furthermore, the maintenance of relative nucleic acid integrity was evaluated on histological sections by using in situ hybridization.
The formulation of GAF® initially tested for terrestrial species exhibited suboptimal results in both species. However, upon adjusting the GAF® formulation for fish species, we observed improved results (see Figure 1 and 2), which more closely resembled those obtained with 10% formalin and 4% paraformaldehyde. Interestingly, Masson’s trichrome staining did not reveal any notable differences among the different fixatives. In GAF®-fixed specimens, in situ hybridization results matched those observed with 10% formalin and the signal was clearer and more pronounced.
In conclusion, GAF® fixative, especially the formulation set up for fish species, could serve as a valuable fixative alternative to formaldehyde for both freshwater and marine fish species.