Evaluation of autofluorescence quenching techniques on formalin- fixed chicken tissues

Abstract

Autofluorescence (AF) in formalin-fixed and paraffin-embedded tissues limit their use in immunofluorescence staining techniques. Various methods have been used to reduce AF in human and animal tissues but no protocol has been optimized for avian tissues. The present study was undertaken to evaluate different treatment methods including ammonium chloride, glycine, Trypan blue, sodium borohydride, Sudan Black B, potassium permanganate, LED light, cupric sulphate combined with glycine, ammonium chloride and cupric sulphate in reducing AF in FFPE chicken tissues for the detection of FITC labelled antibodies against immune cell markers. Chicken tissues including conjunctiva, trachea and Harderian gland presented intense non-homogenous AF in cells resembling erythrocytes, connective cells and melanocytes. Only Sudan Black B effectively reduced AF in FFPE tissues; however, no specific fluorescent signal was observed for six FITC labelled antibodies against immune cell markers. Specific fluorescent signal from the FITC-labelled antibodies was observed in frozen chicken tissue sections with minimal AF, suggesting that the AF in FFPE tissues is related to the use of formaldehyde fixatives. In conclusion, this study demonstrates for the first time that AF quenching methods commonly used for other animal species are not appropriate for use in avian tissues and that frozen tissue sections are recommended for immunofluorescence staining techniques in poultry.