Fixation on Histology

Troubleshooting Fixation in Histology Pt. 1: Expert Tips for Smooth Sailing


Whether you're a seasoned histotech or a newbie, we've all been there – those moments when your samples just don't quite fix the way they should. But fear not, because here are some tips to help you sail through those rough waters and ensure your histological specimens are perfectly preserved.

The Basics of Fixation

Before we dive into troubleshooting, let's quickly recap the basics of fixation. Fixation is the process of preserving tissue samples by chemically stabilizing their cellular structures. Proper fixation is essential to ensure that the tissue's morphology and molecular components remain intact for downstream processing and analysis.

Fixation’s primary purpose is preservation.  It prevents autolysis (self-digestion) and putrefaction of the tissue, keeping it in a suitable condition for analysis.  In addition most fixatives cross-link proteins, helping to maintain the structural integrity of cells and tissues.

Common fixatives include formalin, paraformaldehyde, alcohol-based fixatives, and a range of specialty fixatives tailored for specific applications. However, despite using appropriate fixatives, things can still go awry. So, let's roll up our sleeves and get ready to troubleshoot!

Problem: Over-Fixation

Symptoms: Tissue becomes rigid, difficult to section, or exhibits poor staining.

Causes: Excessive fixation time or use of strong fixatives.

Solution: For those of us who tend to be a bit overzealous with fixation, remember that "more" isn't always "better." Reduce fixation time or switch to a milder fixative if appropriate for your study. Always follow the recommended fixation times for specific tissues and fixatives.

Problem: Under-Fixation

Symptoms: Tissue is fragile, cells are distorted, or nuclear detail is compromised.

Causes: Insufficient fixation time or use of weak fixatives.

Solution: Be patient – under-fixation is often the result of not allowing enough time for the fixative to penetrate the tissue adequately. If you're dealing with larger specimens, consider sectioning them into smaller pieces or increasing the fixation time. Also, ensure you're using an appropriate fixative for the type of tissue you're working with.

Problem: Fixative Incompatibility

Symptoms: Tissue turns black, white, or exhibits unusual color changes.

Causes: Mixing incompatible fixatives, using a fixative not suited for your sample, or failing to properly neutralize acidic fixatives.

Solution: Always ensure compatibility between your fixatives. If you need to switch between fixatives during processing, make sure you neutralize acidic fixatives with an appropriate buffer solution. And remember, the right fixative for the right job is crucial to avoid unusual color changes.

Want to learn more about fixation?  Check out this free NSH member webinar – Fix It or Forget It!!!

Get ready for Part 2, where we dive into advanced troubleshooting and pro tips for perfectly preserved tissue samples.