Streptavidin alexa 350

After 1 week of tachyzoite conversion as described above, in vitro cysts were fixed and stained with biotinylated dolichos (primary, 1:400; Vector Laboratories) and streptavidin Alexa 350 (secondary, 1:1,000; Life Technologies). ImageJ was used to select dolichos-positive cysts and quantify the total intensity of tdTomato-Atg8 and the tdTomato-Atg8 puncta size within each cyst. Dolichos-positive structures between 200 and 2,000 μm²with a circularity of 0.40 to 1.00 were identified using the minimum method of autothresholding. The resulting binary images were used to create masks under which Atg8 puncta were further analyzed. tdTomato-Atg8 puncta were analyzed as being between 0.2 and 1.50 μm² with a circularity of 0.40 to 1.00 and were identified according to the Phansalkar method of autolocal thresholding with a radius of 15 (27 ).

Tachyzoites were converted to bradyzoite cysts as described above. After 7 days of conversion, parasites were treated with 1 μM LHVS or DMSO (control) every day for 3 days. The cells were then fixed and stained with biotinylated dolichos lectin (primary, 1:400; Vector Laboratories) and streptavidin Alexa 350 (secondary, 1:1,000; Life Technologies). ImageJ was used to select dolichos-stained cysts and quantify the number and size of puncta within the cyst. Images were automatically thresholded using the MaxEntropy method to create a binary image (28 (link)). Noise was reduced by opening the image with six iterations of one pixel. Masks were created by using the “analyze particle” function, with objects between 130 and 1,900 μm² and a circularity of 0.30 to 1.00 being called a cyst. Under these masks, dark puncta were analyzed as follows. Phase images were Gaussian blurred with a sigma of 2 and then autolocal thresholding was performed according to the Phansalkar method (27 ) with a radius of 5 pixels. Objects with an area of 0.20 to 6.00 μm² and a circularity of 0.50 to 1.00 were analyzed as dark puncta.

After 1 and 2 weeks of tachyzoite conversion, as described above, in vitro cysts were fixed and stained with biotinylated dolichos (primary, 1:400; Vector Laboratories) and streptavidin Alexa 350 (secondary; 1:1,000; Life Technologies). ImageJ was used to select dolichos-stained cysts and quantify the amount of GFP coverage and intensity within the cyst. The dolichos signal was used to create a mask for further analysis by autothresholding according to the method of Li and Tam (26 (link)). Analysis under these masks was redirected to the GFP channel, where particles of between 130 to 2,300 μm² and 0.30 to 1.00 circularity were analyzed.