Which fixation method is commonly associated with increased autofluorescence that can be mitigated by a specific treatment?

Which fixation method is commonly associated with increased autofluorescence that can be mitigated by a specific treatment?

• A. Aldehyde fixation increases autofluorescence; mitigate with 0.1% sodium borohydride in PBS
• B. Alcohol fixation increases autofluorescence; mitigate with ethanol wash
• C. Heat fixation increases autofluorescence; mitigate with cooling
• D. No fixation affects autofluorescence

Answer: (A)

Aldehyde fixation—using agents like formaldehyde or glutaraldehyde—tends to boost autofluorescence because the crosslinking and formation of Schiff base–type adducts create fluorescent compounds in the tissue. Treating with sodium borohydride in PBS reduces these carbonyl groups, converting them to less reactive alcohols and thus markedly lowering the unwanted fluorescence. The commonly used concentration is around 0.1% NaBH4 in PBS, applied for a short period with thorough washing afterward. This approach specifically targets the aldehyde-induced autofluorescence; other fixation methods don’t have a reliable, standard quencher like this, and a simple wash or cooling does not address the underlying autofluorescent species.

Aldehyde fixation—using agents like formaldehyde or glutaraldehyde—tends to boost autofluorescence because the crosslinking and formation of Schiff base–type adducts create fluorescent compounds in the tissue. Treating with sodium borohydride in PBS reduces these carbonyl groups, converting them to less reactive alcohols and thus markedly lowering the unwanted fluorescence. The commonly used concentration is around 0.1% NaBH4 in PBS, applied for a short period with thorough washing afterward. This approach specifically targets the aldehyde-induced autofluorescence; other fixation methods don’t have a reliable, standard quencher like this, and a simple wash or cooling does not address the underlying autofluorescent species.