Abstract
Formaldehyde fixation is the main method for crosslinking cellular proteins prior to their usage in immunocytochemistry. In order to create these links, formaldehyde undergoes a Mannich reaction in which the formaldehyde forms a methylene bridge between the aminogroup of two amino acids. Crosslinking increases protein stability allowing for more accurate preservation of in vivo conformations which in turn increases binding affinity of fluorochrome conjugated antibodies for fluorescent imaging. Formaldehyde is also a known carcinogen as classified by the National Cancer Institute. Malonic acid, a green, plant-based, water-soluble, and relatively inexpensive polycarboxylic acid has been shown to undergo crosslinking of proteins through an unknown mechanism. To test whether malonic acid can crosslink proteins within cells we fixed SH-5YSY cells with either malonic acid or formaldehyde and then stained with a fluorochrome conjugated antibody for the cytoskeletal protein α-tubulin. The cells were then imaged 72 hours after fixation. We observed a non-significant difference in the fluorescence of immunostained SH-5YSY cells fixed with malonic acid as compared to paraformaldehyde (p-value = 0.2469, ANOVA). In addition, we have created a theoretical mechanism showing malonic acid forming a propyl bridge for crosslinking proteins in a similar mechanism to that of formaldehyde. Here, we show that malonic acid is able to fix cells and retain fluorescence just as well as paraformaldehyde up to 72 hours after fixation and present several possible mechanisms for this chemical process.
Highlights
In order to test the preservation capabilities of malonic acid compared to paraformaldehyde after immunostaining, we imaged unconjugated monoclonal mouse anti-tubulin primary antibody with TRITC-conjugated donkey anti-mouse secondary antibody 72 hours after the fixation process
We found that 72 hours after fixation, malonic acid fixation (Figure 2(b)) was comparable to that of formaldehyde (Figure 2(a)) for the staining the staining of SH-SY5Y cells for tubulin
The corrected total cell fluorescence shows a similarity in the overall fluorescence of the two groups (Figure 3) with statistically insignificant differences using an ANOVA (n = 6, DF = 1, F = 1.46, p-value = 0.2469)
Summary
Cellular fixation is an important step for the use of processing monolayers of cells during immunocytochemistry. Fixation helps preserve the overall cytoskeletal use of additional processing. Fixation has been shown to essentially freeze proteins and other biological molecules in their spatial locations within the cell [1] [2]. Formaldehyde can combine with nitrogen and some other atoms of proteins or with two such atoms if they are very close together, forming a crosslink -CH2- called a methylene bridge. Studies of the fixation of collagen have shown that the most frequent type of crosslink formed by formaldehyde in collagen is between the nitrogen atom at the end of the side-chain of lysine and the nitrogen atom of a peptide linkage [5]
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