Abstract
Background:
Formalin is a widely used histological fixative despite its carcinogenic properties, inadequate nucleic acids preservation, and prolonged fixation time.

Aim:
The study intended to prepare a novel zinc-based fixative which is, formalin-free, and cost-effective, provide optimal preservation, with rapid penetration rates, and enhanced Hematoxylin/Eosin-stained sections.

Methods:
Liver, kidney, brain, and small intestine specimens were collected from ten adult laboratory rats (Rattus norvegicus) and preserved in formalin 4%, formalin 10%, NBF and the experimental EZAT solution. The penetration depth (discolored edges) was measured at 6, 12, 24, and 48-hours. The shrinkage ratio was calculated by measuring the width and length of the samples before and after 48 hours of fixation. The histological evaluation, included staining affinity, cellular outline, cytoplasmic and nuclear attributes, and overall tissue structure, was conducted by a panel of academic experts and rated using a scale of poor (1), fair (2), good (3), very good (4), and excellent (5) grades. The data were later statistically analyzed to determine the significant differences among the tested fixative types.

Results:
Higher penetration rate was observed with the EZAT solution at 6 and 12 hours’ time and the samples were reached at optimal fixation after 24 hours; with an accelerated diffusion coefficient, and a minimal shrinking effect compared to formalin 10% and NBF. The microscopic evaluation of Hematoxylin/Eosin-stained sections revealed better staining affinity, refined histological details, and enhanced cytoplasmic and nuclear properties. The overall structural evaluation revealed an excellent microscopic appearance with the EZAT solution compared to formalin-based fixatives.

Conclusion:
The EZAT fixative should be considered as an everyday preservative in histology and histopathology laboratories. Future studies should be focused on the potential of EZAT in cellular histochemistry and immunohistochemistry practices.

Key words: Formalin, Histology, Laboratory rats, Zinc-based fixative