Methylene bridges are formed during fixation by formaldehyde linking which groups?

• A. OH groups
• B. NH2 groups
• C. COOH groups
• D. SH groups

Answer: (B)

Formaldehyde fixes tissue by creating methylene bridges between protein molecules, locking their structure in place. It primarily targets amino groups—the free amine sites on proteins, especially the N-termini and the side chains of lysine residues. The mechanism starts when formaldehyde reacts with a primary amine to form a hydroxymethyl adduct. A second amine can then attack this intermediate, releasing water and forming a -CH2- bridge that links the two nitrogen atoms. This crosslinking between amino groups is what preserves cellular architecture during embedding and processing. Other functional groups like OH, COOH, or SH are not the main sites for forming these methylene crosslinks under standard fixation conditions, which is why amino (NH2) groups are responsible for the methylene bridges.