Fixation and Shipment of Wet Tissues for Paraffin Embedding Downstream Services

Experimental tissue samples are important to any study, so before you sacrifice your animal or start preparing buffers, here a few fundamental techniques and considerations that will help optimize your results before sending those samples to us for further histopathological analysis.

Choosing Your Fixative
The type of fixative used is determined by the specific experimental goals, tissue type, and downstream applications. Formalin is generally recommended and is the most used fixative for histopathology or immunohistochemistry (IHC) samples. Ethanol is a good fixative for preserving lipids and cellular membranes for histopathology but not for immunohistochemistry (IHC) samples. Methanol is a commonly used fixative for frozen sections and immunofluorescence (IF) and immunohistochemistry (IHC) assays. Bouin’s fixative is used for preserving glycogen and elastic fibers and is for histopathology samples only. Acetone is commonly used for preserving lipids and cellular membranes used for frozen or cell cultures for immunohistochemistry (IHC) and immunofluorescence (IF) assays.

Fixation Process
While there are different methods of fixation of various situations, the two most commonly used are perfusion fixation and immersion fixation.
Perfusion fixation is a method of preserving biological tissues for further study or experimentation. The fixative solution is injected directly into the bloodstream by cardiac or vena cava puncture, allowing the fixative to permeate the tissues and preserve their structure and molecular content.
Immersion fixation involves immersing the tissue in fixative solution. Since the solution should completely diffuse through the sample, the size and density of the sample should be a major consideration. For best results, the sample should be immersed in the appropriate fixative at a minimum volume of 20 times greater than the sample.
The amount of fixative needed for histopathology samples is determined by the size and type of tissue being studied, as well as the desired level of fixation. To ensure adequate penetration of the fixative solution, the volume of fixative should be at least 20 times the volume of the tissue to be fixed.
Perfusion fixation may be required for larger tissues, such as organs or whole bodies, to ensure complete fixation.

Working Quickly
Prompt fixation is critical to preventing tissue sample degradation caused by autolysis and other factors.
Tissues should be fixed as soon as possible after being collected, ideally within minutes. The longer the time between tissue collection and fixation, the greater the risk of tissue damage on cellular morphology, which can jeopardise histopathological analysis's accuracy and reliability.

Duration of Fixation
Over fixation can cause tissue hardening and antigenic site masking, while under fixation can result in poor cellular structure preservation. The amount of fixation time will vary depending on the type of tissue being studied and the specific experimental goals.
The length of fixation should be sufficient for histopathology to ensure optimal preservation of tissue morphology and cellular structures. Formalin fixation is commonly used in histopathology, with fixation times ranging from several hours to overnight depending on the size and type of tissue being studied.
For IHC, the fixation time should be long enough to preserve antigenicity while also preserving tissue morphology. Shorter fixation times are generally preferred for IHC to preserve antigenicity. Fixation times of 6-24 hours (maximum 48 hrs) are commonly used for IHC using formalin-fixed paraffin-embedded (FFPE) tissue to preserve both tissue morphology and antigenicity.

Storage and Shipping Considerations
Important considerations should be made when shipping wets to our lab to ensure that they are not damaged during transit. In general, samples can be shipped in formalin for all subsequent downstream procedures. Wet samples sent for OCT downstream services with immunohistochemistry (IHC) or immunofluorescence (IF) can be shipped in neutral phosphate buffered saline (PBS) depending on timelines. Wet samples to be processed for paraffin embedding can also be shipped in 70% ethanol and can also be used for long term storage.
When packing samples for shipment, the type of sample, the required temperature conditions, and the shipping method must all be carefully considered when shipping and packaging histopathology and IHC samples. Proper sample labelling, documentation, packaging, and cushioning can help ensure that the samples arrive in good condition and on time for further analysis.
For more suggestions or advice on how to best collect and fix your tissue samples, contact or read our fixation protocol for more in depth details.

Wax-it Histology Services Inc.