Lsm 880 upright laser scanning confocal microscope

To quantify LBP-2::tagRFP aggregation in the tail region of C. elegans, age-synchronised nematodes at indicated time points were immobilised in 10 mM sodium azide on 2% agarose (w/v) pads on a microscope slide. Images were captured on a LSM880 upright laser scanning confocal microscope (Zeiss, Oberkochen, Germany) with a 40 × oil objective, using the Zen software. tagRFP was detected using a 561 nm excitation laser and an emission range from 565–650 nm. Representative confocal images are displayed as maximum z-stack projections.
For aggregation quantification, the tail region was defined as the region from the tip of the tail to the anal sphincter. We chose the tail region because of interference of the myo-2p::mCherry co-injection marker in the head region of β₂m and ctrl animals. Aggregates were quantified in a semi-automated process. ImageJ was used to subtract the background and apply a threshold to a maximum intensity projection images. Aggregates were defined as fluorescent puncta when larger than 1 µm². Two independent experiments were performed for each strain (n = 10–21 worms per condition per experiment) and error bars indicate S.E.M. For statistical significance, a two-way ANOVA followed by a post-hoc Tukey’s test was performed.