Six Common Mistakes to Avoid When Preparing Samples for a Histology Study

Histology is a crucial tool for examining changes in tissue morphology or microstructure when studying diseases or disease models. However, preparing specimens for paraffin processing involves several steps, and errors at any of these stages can significantly impact the quality of the final staining. Some mistakes can even be catastrophic. Below are some common mistakes we've observed, which you should aim to avoid:

1. Using a Regular Laboratory Marker for Specimen Labeling
   Although this may not be the most frequent mistake, it can have the most severe consequences. Many histology core facilities still use xylene as a clearing agent during paraffin processing. While regular laboratory markers, such as Sharpies, may be suitable for general labeling, they are not resistant to xylene. Consequently, labels on cassettes marked with these pens can be completely washed off during processing, leading to the total loss of sample identity. We always recommend using histology-grade markers or a pencil for labeling cassettes if a cassette printer is unavailable.
2. Incomplete Tissue Fixation
   Incomplete fixation occurs when part of the tissue, typically at the center, is not fully fixed. Tissues for paraffin processing usually require 24 to 72 hours of fixation before being transferred to 70% ethanol or PBS. Incomplete fixation can result from insufficient fixation time, overly thick tissue, or the presence of a tissue capsule that prevents the fixative from penetrating. This can negatively impact downstream sectioning and staining.
3. Delayed Tissue Fixation
   Delayed fixation happens when tissue is not placed in fixative in time, leading to tissue partial autolysis before complete fixation. This can affect subsequent staining and introduce artifacts that complicate data interpretation.
4. Over-fixation of Tissue
   While incomplete or delayed fixation can harm sample quality, over-fixation can also be problematic, particularly for immunohistochemistry (IHC) staining. While most chemical stains can tolerate longer fixation times, over-fixation should be avoided if you plan to conduct IHC, especially when using fluorescence detection.
5. Issues During Paraffin Processing
   The steps of paraffin processing, such as tissue dehydration, clearing, and paraffin infiltration, are essential for achieving the processing quality. These procedures are typically carried out in a histology core lab. It's important to keep chemicals fresh and use custom protocols tailored to different tissue sizes or types to ensure optimal tissue processing.
6. Embedding Large and Small Tissues in the Same Block
   Unlike clinical settings where tissues are typically embedded individually, researchers often embed multiple tissues in one block to enhance consistency and efficiency. However, this practice can reduce your flexibility in selectively staining specific tissues without affecting others. If small tissues are embedded alongside larger ones, the smaller tissues might be entirely trimmed off when attempting to expose the larger ones. Therefore, it's advisable to embed smaller and larger tissues separately.