Tissue Fixation Problems: How to Prevent Formalin Pigments & Under-Fixation

Fixation and grossing form the foundation of the entire sectioning workflow — get these two stages wrong, and every downstream step compounds the error.

Phase 1: Fixation and Grossing

The primary fixatives utilized for tissue sectioning are formaldehyde and alcohol. Fixatives cross-link and denature proteins, thereby hardening the tissue and stabilizing its structure. Furthermore, the denatured substances can bind with dyes, enhancing the staining capability. Simultaneously, fixation precipitates and cross-links intracellular components such as proteins, lipids, carbohydrates, and enzymes. This creates distinct refractive indices, making the fine structures of cells and tissues easier to observe clearly under an optical microscope.

1.1 Reducing or Eliminating Formalin Pigments in Tissue Sections

Saturated formalin solution is slightly acidic, which adversely affects nuclear staining. Notably, after prolonged storage, it tends to precipitate and deposit within tissue sections as fine, dark brown or yellowish-black granules (formalin pigments), which interfere with microscopic observation.

Countermeasures:

• Standard Solution: A 10% neutral buffered formalin (NBF) solution, prepared by mixing saturated formalin with pH 7.2 PBS buffer at a 1:9 volume ratio, is the most widely used and effective method to reduce and eliminate formalin pigments.

• Field Alternative: If PBS buffer cannot be prepared on-site, ordinary white chalk can be added to the fixative solution to maintain a neutral or weakly alkaline pH.

• Note: Formalin pigments frequently appear in areas with high blood and plasma distribution, as well as in severely decomposed tissues. Consequently, they can serve as an indicator of tissue hemorrhage and plasma exudation.

1.2 Preventing Poor Tissue Fixation (Under-fixation)

If the fixative is too concentrated or too dilute, its ability to penetrate tissue diminishes, leading to uneven fixation and under-fixed core zones. This not only results in uneven tissue hardness and uneven cut surfaces during grossing—which hinders the trimming of the block face during sectioning—but also compromises subsequent dehydration, wax infiltration, and staining outcomes.

Countermeasures:

• Volume Ratio & Time: Generally, the optimal volume ratio of fixative to specimen is approximately 10:1. Organs and tissues must be completely submerged in the fixative for 3 to 7 days. For severely autolyzed or decomposed specimens, the fixation time should be extended by a few days, or the formaldehyde concentration should be increased (e.g., prepared at a 1:8 ratio). The fixative concentration should also be appropriately increased in cold environments.

• Organ-Specific Protocols: Fixative penetrates tissue at a rate of about 2 to 3 mm per day. To ensure rapid and complete fixation, larger organs such as the brain, liver, lungs, heart, kidneys, and spleen must be incised before fixation.

  • Whole Brain: Should be suspended with a cord for fixation. For routine pathological examination, making a median sagittal cut through the corpus callosum prior to fixation can accelerate the process.

  • Heart: All atrial and ventricular cavities should be routinely opened.

  • Lungs & Kidneys: A single cut should be made from the maximum outer margin toward the hilum.

  • Liver & Spleen: Routinely sliced parallel to the long axis into 2–3 cm thick slabs before fixation.

  • Maintenance: If the organ is large, the container is small, or the volume of fixative is insufficient, the organ should be turned over or the fixative replaced within the first 3 days to prevent the surface resting against the container wall from flattening or becoming under-fixed.

1.3 Avoiding Uneven Thickness and Irregular Cavities in Tissue Sections

Grossing is generally performed 3 to 7 days post-dissection. During grossing, the prosector must use a sharp knife and a steady hand to ensure flat cut surfaces and a uniform thickness of 3 to 5 mm. This prevents uneven block thickness and irregular observation surfaces.

Countermeasures:

• Block Size: The size of the tissue block should be 1 to 2 mm smaller than the selected coverslip to ensure complete mounting and sealing.

• Labeling: Tissue labels must be made of durable paper and marked with insoluble carbon-based ink to prevent the text from being dissolved or faded during dehydration and clearing.

• Washing: Tissue blocks should be thoroughly washed under running tap water for at least 12 hours to rinse away and dilute residual formaldehyde solutes and particulates.

  **Part of our Tissue Sectioning Troubleshooting series.** See the [complete 9-step guide]or jump to another phase: [Fixation & Grossing] | [Dehydration & Embedding]| [Sectioning & Floating]