For most tissues, in vivo perfusion fixation of tissue produces superior tissue morphology compared to immersion fixation. Depending on the tissue being collected and the experimental animal used, specific perfusion protocols are available. In vivo perfusion-fixation removes blood from the tissues during a pre-fixation rinse and allows rapid fixation of cells and retention of tissue architecture by delivering the fixative directly while maintaining open vessels, open lumens, and interstitial spaces.
Aside from doing in vivo perfusion, the choice of fixative is extremely important. For immunolocalization studies by light microscopy or electron microscopy, we typically recommend 2 to 4% paraformaldehyde-lysine-periodate (PLP) as the primary fixative; in the study featured on the JASN cover, the Wall laboratory preserved the mouse kidneys by in vivo cardiac perfusion with 3% PLP after a PBS rinse.
PLP fixative was developed by McLean and Nakane (1) as a fixative for ultrastructural immunolocalization to improve the signal:noise in immunogold labeling, and we find it excellent for immunohistochemistry and immunofluorescence, as well. PLP stabilizes polysaccharide chains through lysine cross-links, which helps in preserving structure and antigenicity. Preparing the fixative requires several steps: separate buffers (lysine, monobasic and dibasic sodium phosphate) and paraformaldehyde solution are made; paraformaldehyde is made no sooner than 24hrs before use. On the day of fixation, we combine the separate buffers, mix with the paraformaldehyde solution, and finally add periodate. Paraformaldehyde oxidizes to form formic acid, with a corresponding steady drop of pH and osmolarity over time. So, to avoid degradation of paraformaldehyde, the working fixative solution should be made immediately before use.
10% neutral buffered formalin is a common commercial fix that many people use, it is comparable to 3.7% paraformaldehyde. However, formic acid is a common contaminant of commercial or stored formaldehyde solution, and based on Fox et al.’s findings, a freshly made formaldehyde solution after a month of storage yields about ten times less formic acid than month storage of commercial 10% buffered formalin (2). Although formic acid has a small effect on cell nuclear size, it does present a birefringent pigment precipitate in the tissue when blood-rich tissues are fixed in formaldehyde containing excess formic acid (2).
Using a fixative with too high or too low osmolarity can have an adverse effect on the morphology of the tissue. Thus the osmolarity of the buffer used for rinsing and as a base for the fixative should be near the ambient osmolarity of the tissue being collected. Aldehydes (paraformaldehyde, glutaraldehyde) add to the measured osmolarity of the fixative solution, but because they cross cell membranes freely, they do not add to the effective osmolarity in the tissue. Our formulation gives good morphology of the renal cortex and outer stripe of the outer medulla. To avoid using fixative that was mixed incorrectly or with a bad reagent, we always check and record the final pH and osmolarity of the finished fixative to make sure that they are within the expected and desired range before continuing the experiment. We use a μOsmette osmometer (Precision Systems, Inc., Natick, MA) to measure our solution osmolarity. The measurement is by freezing point depression; we have found that vapor pressure osmometers do not read aldehyde solutions correctly. Per-user requests, we offer service for osmometer use.
- McLean IW, Nakane PK. (1974). Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 22(12):1077‐1083.
- Fox CH, Johnson FB, Whiting J, Roller PP (1985). Formaldehyde fixation. J Histochem Cytochem. 33(8):845‐853.