Efficacy of Sodium Borohydride for Autofluorescence Reduction in Formalin-Fixed Paraffin-Embedded Common Quail (Coturnix coturnix) Lungs Under Different Antigen Retrieval Conditions

Tissue autofluorescence (AF) is a major limitation to immunofluorescence (IF) staining of formalin-fixed paraffin-embedded (FFPE) tissues. This study evaluated the AF reduction efficacy of sodium borohydride (NaBH₄) in FFPE quail (Coturnix coturnix) lung tissues processed under heat-induced epitope retrieval (HIER) or enzyme-induced antigen retrieval (EIAR) conditions. Following antigen retrieval (AR), lung sections were treated with 1 or 2 mg/mL NaBH₄ concentrations and compared with untreated controls. Autofluorescence was evaluated across green and red channels under identical imaging settings; and mean fluorescence intensity (MFI) was quantified using Fiji (ImageJ). Data were analyzed by one-way ANOVA with Tukey's post hoc test. NaBH₄ significantly reduced AF in green and red channels (p < 0.05) under both AR conditions. No difference was observed between 1 and 2 mg/mL treatments. Collagen type-IV were detected in HIER-processed tissues but not in the EIAR-processed ones. These observations shows that NaBH₄ is effective in reducing green and red AF in FFPE quail lungs regardless of AR method employed; and immunostaining of collagen type-IV in FFPE quail lung is better achieved with HIER than with EIAR methods. This study presents an optimized approach for reducing AF and enhancing antigen visualization in IF studies of avian FFPE lung tissues.