Unveiling the Secrets of Fixation

August 14, 2023
Author: Anisha Bhola
Editor: Dr. Jitendra Kumar Sinha

Name of Technique: Fixation of the neural tissue. 

Used along with: Histology, Immunohistochemistry, In situ Hybridization, Electron Microscopy, Molecular Studies, and Tissue Banking or Archiving. 

Purpose: Tissue preservation, provide cellular and subcellular details, Molecular analysis, Long-term storage, comparison and standardization.

Figure 1 – Fixed Human brain displayed for demonstration (Photo by Anisha) 

To effectively examine the structure of neural tissue, researchers must maintain its natural state as much as possible due to its soft, delicate, and degradable nature. Therefore, the primary objective of an investigator is to preserve the tissue’s living characteristics accurately. This is achieved through a series of steps that involve fixing, embedding, and sectioning the tissue before employing visualization techniques. Fixing and embedding processes are utilized to stabilize the tissue and retain its current condition, while sectioning enables the tissue to be thinly sliced, allowing light to pass through and facilitating the examination of internal structures using a microscope. 

Meticulous attention is essential to achieve a comprehensive understanding of the brain and the intricate workings of the nervous system. In this regard, fixation plays a crucial role.Fixation is the process by which neural tissues are preserved and stabilized. It involves rendering the tissues and cells in a physically and chemically stable state. Fixation has a significant impact on our understanding of the brain as it provides insights into its biological functioning and structure. 

The primary purpose of fixation is to ensure that the tissue can withstand subsequent treatments with minimal loss or decomposition. For histological examinations, proper fixation is of utmost importance, as the absence of fixation would render any experiment conducted ineffective.When tissues are not fixed, the accumulation of CO2 and metabolites increases, leading to oxygen deprivation and subsequent tissue decomposition. Fixation is performed to prevent putrefaction (the process of decay) and autolysis (the action of various enzymes) from occurring.Therefore, fixation is carried out to preserve the structural integrity of the tissue in a manner that closely resembles its natural state. 

There are two methods by which fixation can be done: 

  1. Physical methods like heating, microwaving, and freeze-drying. 
  1. Chemical methods: Adequate morphological preventions are maintained by using Non-organic and organic solutions as chemical fixatives. Compound, cross-linking, and coagulant fixatives are three major categories of chemical fixative. 
Figure 2 – Representative images of brains dissected out under different conditions [Sourced fromWu J et al., 2021]

When tissue is fixed, the fixatives precipitate or denature the proteins because of which a cross-linking of protein happens and a meshwork is formed. The cells hold on to each other in an in vivo condition due to this cross-linking of proteins. The mechanical strength which is needed for subsequent procedures is obtained with the soluble proteins. 

  1. Freshly dissected brain is full of blood and very soft.  
  1. Unsuccessfully perfused brain retains some blood.  
  1. Successfully perfused and fixed brain before post-fixation. 
  1. Brain after post-fixation. 
Figure 3 – Flow chart representing fixation and embedding methods 

Fixatives preserves the structural integrity of neural tissue: 

Consider a situation where you are trying to study the brain without appropriate fixation. This would lead to putrefaction and rapid deterioration of the tissue which in turn lead to the loss of vital information for future experiments. 
 

Fixation maintains structural integrity and acts as a guardian. Postmortem changes are prevented with the help of fixation as by halting enzymatic processes the tissue retains its natural form which makes it easier for researchers to examine them under a microscope. Due to enzymatic inactivation, cellular component degradation is prevented and molecular composition prevention helps in researching the intricate neuronal functioning details. Many signaling pathways and molecular interactions are explored as the fixation of tissue provides the researchers with snapshots of the biochemical landscape. 

Ideal fixative’s Properties: 

  1. It must be safe to use, cheap, and stable (Safety, Affordability, and Stability: The Triad of Excellence). 
  1. It should not alter the chemical and physical state of the tissue and its components (Preservation of Chemical and Physical Integrity). 
  1. It should penetrate the tissue quickly (Expeditious Tissue Penetration: A Matter of Efficiency). 
  1. Fixatives should be rapid in action (Rapid Action: Unveiling the Latent Potential).  
  1. It should cause minimum loss to the tissue (Minimization of Tissue Loss: Maximizing Investigative Yield). 
  1. It should be isotonic (The Balancing Act: Achieving Isotonicity). 

Choosing the right fixative: 

The techniques which are supposed to be employed along with the research objective play a major role in choosing the right fixative. 

For instance: 

  • Glutaraldehyde is used to preserve the ultrastructure of the brain for Electron microscopy. 
  • Paraformaldehyde is used to preserve brain tissue for cryo-sectioning and immunohistochemistry studies. 

Factors affecting fixation: 

  1. Concentration of fixative plays a very major role. 
  1. If pH is not between 6 to 8, it may alter the process of fixation. 
  1. Slow penetration of fixative happens when tissue is covered by blood or mucus. 
  1. Increased fat content in a tissue may result in slow fixation. 
  1. Room temperature is the most favourable temperature of fixation. 

Paraformaldehyde fixation protocol: 

Wu, J. et al. (2021) Transcardiac perfusion of the mouse for brain tissue dissection and fixation, Bio-protocol. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005872/ (Accessed: 11 July 2023).  

Conclusion: 

Fixation is an indispensable step in neuroscience research, laying the groundwork for further investigations into the brain and nervous system. By preserving tissue structure, inactivating enzymes, preventing degradation, and enabling detailed molecular studies, fixation allows researchers to unlock the mysteries of the brain. With its ability to freeze neural tissues in time, fixation provides the stability necessary for accurate analysis and a deeper understanding of the complexities of neural architecture and function. 

References: 

  • Bancroft, J. D., & Gamble, M. (2014). Theory and Practice of Histological Techniques (7th Edition). Churchill Livingstone. 
  • Carter, M., & Shieh, J. (2015). Guide to Research Techniques in Neuroscience (2nd Edition). Academic Press. 
  • Wu J, Cai Y, Wu X, Ying Y, Tai Y, He M. Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation. Bio Protoc. 2021 Mar 5;11(5):e3988. doi: 10.21769/BioProtoc.3988. PMID: 33796622; PMCID: PMC8005872.