Description: Formalin is a commonly used fixative for tissue preservation in histopathology labs. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking through formaldehyde reaction with the protein amino groups. Citraconylation by citraconic anhydride provides an easy method for the reversible blocking of protein amino groups. The method provides efficient AR for successful immunostaining of a wide variety of antigens under an optimized condition.
Solutions and Reagents:
0.05% Citraconic Anhydride Buffer (pH 7.4):
Citraconic anhydride ------------------ 0.5 g
Distilled water --------------------- --- 1000 ml
Mix to dissolve. Adjust pH to 7.4
Deparaffinize sections in 2 changes of xylene, 5 minutes each.
Pre-heat steamer or water bath with staining dish containing
Citraconic Anhydride Buffer until temperature reaches 95-100
Rinse sections in PBS Tween 20 for 2x2 min.
Block sections with for 30 minutes.
Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
Rinse sections with PBS Tween 20 for 2x2 min.
Block sections with peroxidase blocking solution for 10 minutes.
Proceed to standard immunohistochemistry protocol.
1. Namimatsu S, Ghazizadeh M and Sugisaki Y (2005) Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem. 53(1):3-11. PubMed Abstract