Overview: Fixative Solutions

Fixative solutions are liquids that are used to preserve or harden specimens for various purposes in biological research and applications. This is a necessary step in protein, immunology, and cell biology research, as it allows scientists to “freeze” a cell and its components to study it and extract analytes for further review. This prevents cells of interest from changing shape, size, and structure during analysis, and effectively stops further chemical reactions from taking place. Fixation also prevents autolysis and displacement of cell constituents such as antigens and enzymes [2]. Fixative solutions enhance the rigidity and mechanical strength of cells, which is critical to withstanding the various, sometimes rigorous steps of the immunostaining procedure and protects samples against microbial contamination and possible decomposition [3][4]. They are also known to enhance the staining and imaging of biomolecules such as proteins, DNA, RNA, and lipids. Different types of fixatives have different effects on the samples, such as cross-linking proteins, denaturing enzymes, or preserving lipids.

Fixative Solutions Image1

Image 1:Following FUS sonication, an animal brain was harvested upon transcardial perfusion of 4% paraformaldehyde in phosphate buffered saline (BM-155) from Boston Bioproducts prior to analysis [1].

Types of Fixative Solutions

Fixative solutions can vary in composition and are often specific for certain applications. In terms of structures fixed, these solution types often fall under three categories known as Cytologic fixatives, Histochemical fixatives, and Microanatomical fixatives.

Type of Fixative Function Applications
Cytologic Fixatives Fixatives that preserve the cellular components and structures, especially the nucleus Cytology, electron microscopy and chromosome studies
Histochemical Fixatives Fixatives that preserve the chemical constituents of cells or tissues for specific reactions Histochemistry and immunohistochemistry
Microanatomical Fixatives Fixatives that preserve the spatial relationship of tissue layers and large cell aggregations Histology, histopathology, and light microscopy

In each of these fixative category types, fixative solutions can also differentiate in their chemical methods in which they are able to fix a target. These subcategories vary on chemical reactivity, and therefore provide different strengths in experimentation.

Type of Cytologic Fixative Examples Mechanisms Effects and Applications
Cross-linking Fixatives Formaldehyde, Glutaraldehyde Form chemical bonds between adjacent protein molecules Effects: Stabilize cell structure, prevent solubilization, and retain antigenicity Applications: Electron microscopy, immunohistochemistry, in situ hybridization and proteomics
Precipitating Fixatives Ethanol, Methanol, Acetone Remove water from cells, cause protein aggregation

Effects: Harden and shrink cells, make them insoluble, and impair staining Applications: Immunocytochemistry (ICC), flow cytometry, scanning electron microscopy and Fluorescence in situ hybridization (FISH)
Coagulating Fixatives Osmium tetroxide, Picric acid Alter tertiary and quaternary structure of proteins Effects: Make proteins more rigid and resistant, enhance contrast, and distort morphology Applications: Histochemistry, cytochemistry and scanning electron microscopy
Type of Microanatomical Fixative Examples Mechanisms Effects and Applications
Formalin-Based Fixatives Buffered formalin, Neutral buffered formalin, Formol saline Cross-link proteins by forming methylene bridges Effects: Preserve tissue structure and cause minimal distortion, compatible with most stains Applications: Routine histopathology, IHC, antigen retrieval and long-term storage of Formalin-Fixed Paraffin-Embedded (FFPE) blocks.
Mercuric chloride Fixatives Zenker’s fluid, B-5 fluid, Helly’s fluid Precipitate proteins by forming insoluble mercury-protein complexes

Effects: Harden and shrink tissues, enhance nuclear details, impair staining, however, require removal of mercury Applications: Fixation of nuclear chromatin, nucleoli, mitochondria, Golgi apparatus, collagen, glycogen, etc.
Picric acid Fixatives Bouin’s fluid Gendre’s fluid, Hollande’s fluid Coagulate proteins by altering their tertiary and quaternary structure Effects: Swell and soften tissues, enhance cytoplasmic detail, improve staining, however, require removal of picric acid Applications: Fixation of connective tissue, soft specimens, gastrointestinal tract, endocrine glands, glycogen, and other carbohydrates
Alcoholic Fixatives Carnoy’s fluid, Clarke’s fluid, Ethyl alcohol Dehydrate and precipitate proteins by removing water Effects: Harden and shrink tissues, make them brittle, impair staining, and require rehydration Applications: Fixation of cytology specimens, preservation of nucleic acids, improved performance on qPCR assay and biomolecular techniques

Fixative Solutions at Boston BioProducts

Every Fixative Solution is unique to the cell type used and the experimental application. Select the appropriate Fixative Solution from the catalog or design your optimal formulation with custom manufacturing options at Boston BioProducts.

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References:

  1. Yoo, S.-S., Kim, H.-C., Kim, J., Kim, E., Kowsari, K., Van Reet, J., & Yoon, K. (2022). Enhancement of cerebrospinal fluid tracer movement by the application of pulsed transcranial focused ultrasound. Scientific Reports, 12(1), 12940
  2. Adams, J. C. (1992). Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 140(10):1457–1463.
  3. Alelu´-Paz, R., Iturrieta-Zuazo, I., and Byne, W., et al. (2008). A new antigen retrieval technique for human brain tissue. PloS One. 3(10): 3378
  4. Ramos-Vara, J. A. Miller, M. A. (2014). When Tissue Antigens and Antibodies Get Along: Revisiting the Technical Aspects of Immunohistochemistry—The Red, Brown, and Blue Technique. Veterinary Pathology. 51(1): 42-87