Top Highlights

• Indeed, we achieved less background noise using 5% BSA for 60 min at RT as a blocking agent compared to 10% normal goat or donkey sera.
• In line with the recommendation of others, we found that the use of copper sulfate with ammonium chloride in the final rinsing step helped in quenching the autofluorescence effect frequently seen in paraffin-embedded tissues (Schnell et al., 1999). Other protocols described the use of Sudan Black B in ethanol or other reagents to reduce the autoflu...
• In the case that highly vascularized tissues as well as frozen brain sections are used, a series combination of sodium borohydride, crystal violet, and Sudan Black B leads to total quenching of autofluorescence background, masking of lipofuscin, and non-specific signals
• In our hands, 1:400 is the best concentration that works for most secondary antibodies
• if a dilution of 1:300 is recommended, a scientist could test 1:100, 1:300, and 1:500

Domain