Immunostaining of Fungal Hyphae and Fly Hemolymph

The immunostaining protocol was adapted from Luo et al. (29 (link)). F. oxysporum was grown in 5 ml malt extract broth from a starting concentration of 2.9 × 10⁵ spores/ml. After overnight shaking at room temperature, fungal hyphae were collected by centrifugation at 1,000 g for 10 min and resuspended in PBS. Hemolymph was collected via the Zymo-Spin IC column method (23 (link)) from 420 male flies that had been induced with heat-killed M. luteus 24 h prior and incubated at 29°C, yielding ~35 μl cell-free hemolymph. Next, aliquots of 200 μl hyphae and 35 μl hemolymph were shaken at room temperature for 30 min. The samples were washed three times with PBS before fixation with 4% formaldehyde for 1 h. After washing another three times with PBS, samples were blocked for 1 h with 5% BSA. Samples were then incubated with α-FLAG antibody (1:200) overnight at 4°C. After washing with PBS, samples were stained for 2 h with donkey α-mouse Alexa555 (1:400) and DAPI (1:200) and then washed and mounted on slides. Samples were imaged with a Ti2 Widefield microscope (Nikon) and analyzed with the NIS- elements software and OMERO.