Sample Preparation

Fixation for Routine Paraffin Histology

• Functions of a Fixative:

  • Kill the tissue preventing bacterial growth and autolysis preserving the sample as close to living state as possible

  • Denaturation of proteins which renders the proteins insoluble and converts them from their natural gel-like state into a semi-solid

  • Maintain proper relationships between cells and extracellular substances

  • Render tissue more permeable to additional processing reagents

  • Proper fixation enhances staining - poorly fixed tissues stain poorly

• Proper Fixation is the first and most important step for proper preparation of tissue samples - improper fixation cannot be corrected at a later date and can compromise future techniques.  We recommend a project consult with IHP Lab core staff prior to the start of any project.

• Fixative(s):

  • 10% Neutral Buffered Formalin is the most common and widely used fixative for histopathology and may be used for general relationships between cells, tissues and organs. 

    • If focusing on a specific cellular detail, contact the histopathology laboratory for consultation and recommendation of fixative

• Time to Fixative:

  • If possible limit the time of removal from the study subject into the fixative of choice to 20 minutes or less

  • If organs are encapsulated, i.e. kidney - nick or slit the capsule to aid in fixation ~ if possible inflation of lungs via tracheal or gravity perfusion is recommended

• Size of Samples:

  • 2.0 mm to 5.0 mm in thickness (no thicker than a nickel if possible) not to exceed 2.0 cm in circumference

  • Thickness will effect the penetration time of fixatives and other processing reagents

• Volume of Fixative to Sample:

  • Fluid volume should always be 10 - 20 time the volume of fixative to sample size

  • If possible avoid conical tubes as they trap tissue into bottom of tube; not provided access to fixative evenly

• Time in Fixative:

  • Our facility recommends 4 days in fixative - 10% Neutral Buffered Formalin penetrates fairly rapidly 0.5 - 1.0 mm per hour at most;  however it can take days to form aldehyde cross linkages that are stable; with retrieval methods available it is better to over fix than under fix your samples

    • Under fixed tissue can drastically change the antigenicity and ability to retrieve and cause loss of protein content - these losses are permanent

• Temperature:

  • All fixation unless otherwise specified should be carried out at ROOM TEMPERATURE; placing samples at refrigerated temperatures slows down the process of fixation

• Post Fixation:

  • Transfer all samples in cassettes to 30%-70% ethanol for transport to the histology lab