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. This is part two of our Troubleshooting Fixation in Histology blog series.
Problem: Tissue Shrinkage
Symptoms: Tissue samples appear smaller than expected or display irregular shapes.
Causes: Hyperosmolar fixatives or prolonged fixation.
Solution: Opt for isotonic fixatives when possible and limit the fixation time. If shrinkage has already occurred, try rehydrating the tissue with distilled water before further processing.
Problem: Tissue Swelling
Symptoms: Tissue samples appear swollen, and cell morphology is disrupted.
Causes: Hypotonic fixatives or overhydration during processing.
Solution: Use hypertonic fixatives to prevent tissue swelling. If you're dealing with swollen specimens, gently blot them on absorbent paper to reduce excess moisture.
Problem: Inadequate Penetration
Symptoms: Deep layers of tissue display poor fixation, resulting in artifacts.
Causes: Large or thick tissue specimens that weren't adequately trimmed, or rapid fixation methods.
Solution: For larger specimens, slice or trim them to facilitate fixative penetration. Also, ensure you're following the recommended protocols for fixing larger or thicker samples.
Problem: Tissue Adhesion
Symptoms: Sections stick to the microtome blade or slide, causing damage.
Causes: Overfixation, suboptimal tissue processing, or poor sectioning technique.
Solution: Reduce fixation time and use adequate dehydration and clearing steps during processing. Ensure your microtome and blade are clean and well-maintained. Proper sectioning technique is key to avoiding tissue adhesion.
Problem: Artifact Formation
Symptoms: Unintended distortions or imperfections in tissue morphology.
Causes: Overhandling, poor sectioning technique, or contamination during fixation.
Solution: Handle tissues with care, use a well-maintained microtome, and practice good laboratory hygiene. Contamination can often be avoided with clean equipment and meticulous sample preparation.
Problem: Inconsistent Fixation
Symptoms: Tissue samples within the same batch show variations in quality.
Causes: Uneven distribution of fixative, variability in tissue thickness, or inconsistent tissue handling.
Solution: Ensure consistent distribution of fixative by fully immersing your specimens. Maintain uniform tissue thickness during processing and standardize your handling techniques to minimize variability.
Check out this free NSH member webinar, The Basics are Critical: Tissue Fixation, Pre-analytical Variability, and Biomarkers, for more information on troubleshooting fixation.