# A Field‐Friendly, Non‐Toxic Fixative for Integrated Morphological and Molecular Research in Non‐Model Invertebrates

## TL;DR

A new non-toxic fixative called KINFix preserves both tissue structure and RNA in invertebrates collected in the field, enabling combined morphological and molecular studies.

## Contribution

KINFix is a novel, non-toxic fixative that preserves morphology and molecular integrity for multiple downstream analyses in non-model invertebrates.

## Key findings

- KINFix preserves RNA integrity for over 3 months and maintains cellular structure suitable for single-cell RNA sequencing.
- KINFix enables electron microscopy, bulk RNA-seq, and flow cytometry in non-model invertebrates.
- KINFix is a cost-effective and versatile fixative suitable for field-based research with limited specimen availability.

## Abstract

Formalin, ethanol, and RNAlater are the most commonly used fixatives for morphological and molecular studies. Formalin is preferred for preserving tissue morphology, whereas ethanol and RNAlater are used to obtain high‐quality nucleic acids for molecular analyses, including emerging ‐omics techniques. Over the past few years, the study of non‐model organisms has gained attention, but the lack of laboratory cultures for many species requires collecting and fixing the animals directly in the field. Very often, just a few specimens are secured, limiting the possibility of using multiple fixatives for parallel analyses. A single fixative that preserves both morphology and molecules while being easy to handle in the field would therefore be highly valuable. KINFix, a non‐toxic alcohol‐based fixative, was developed to preserve histology, proteins, and nucleic acids simultaneously, enabling both morphological and molecular analyses with the same sample. Here, we evaluate the suitability of KINFix for electron microscopy, RNA preservation, and cell dissociation for single‐cell RNA sequencing (scRNA‐seq) experiments, using four invertebrate species from different spiralian phyla. Our results demonstrate that KINFix maintains RNA integrity for over 3 months, similarly to other standard fixatives, but also preserves morphology and cellular integrity even after cell dissociation, suggesting its suitability for scRNA‐seq applications. While fixation conditions may require optimization for different species and tissues, our findings highlight KINFix as a cost‐effective, versatile, and valuable fixative that enables a wide range of morphological and molecular studies in non‐model invertebrates. KINFix is particularly useful for field‐based research where sample availability and preservation logistics are especially challenging.

Formalin, ethanol, and RNAlater are commonly used fixatives for morphological and molecular studies, but each has limitations when specimen availability is low, as is often the case with field‐collected non‐model organisms. Here, we evaluate KINFix, a non‐toxic alcohol‐based fixative, in four invertebrate taxa and show that it effectively preserves tissue morphology, RNA integrity, and cellular structure, enabling electron microscopy, bulk RNA‐seq, cell dissociation, and flow cytometry analyses, showing potential for scRNA‐seq studies. Our results show that KINFix is a versatile and practical fixative, particularly useful for field‐based integrative morphological and molecular research.