Mounting medium

Sections were deparaffinized and treated with citrate buffer as described above. Subsequently, curcumin staining was performed as described above. After curcumin staining, sections were washed 3 times for 10 min using PBS. Primary antibody was incubated overnight at room temperature. Sections were washed 3 times with PBS and incubated with fluorescently-labelled secondary antibody (1:250, alexa 594, cat# A11037, Invitrogen) for 3 h. After labelling the sections were washed in PBS and covered using DAPI containing mounting medium (cat# 0100–20, Southern Biotech).

After transient transfection with GFP-LC3 or GFP-RFP-LC3, cancer cells were cultured on chamber slide at the concentration of 10³ cells per well overnight. After treatment with bortezomib overnight, cells were fixed with acetone for 5 min and incubated in blocking buffer (5% normal goat serum in PBS) for 1 h at RT to reduce nonspecific binding. For LAMP-2 (Sigma, PRS3627) or cathepsin B (Biovision, 3190-100) staining, cells were incubated with a rabbit polyclonal antibody (1 : 100; Invitrogen, Grand Island, NY, USA; A22283-300L) overnight. After being incubated with Alexa Fluor 546 conjugated anti-rabbit IgG (1 : 100; Invitrogen, A22283-300L), the slides were mounted with mounting medium (SouthernBiotech, Birmingham, AL, USA; 0100-20) and cover glass, and analyzed with the Leica TCS SP2 laser-scanning confocal system (Leica, Wetzlar, Germany) as described previously.^{61 (link)}

Using cercaria or schistosomula isolated as described above and cultured schistosomula, parasites were washed 3 times in ice cold PBS, spinning at 100xg with brakes on the lowest possible setting. They were resuspended in a small amount of PBS and then brought up in 10 mL of 10% neutral buffered formalin and allowed to rotate slowly in the dark at 4°C for 16–24 h. These were washed again 3 times in cold PBS as described above and stored at 4°C until use. For immunostaining, approximately 500 parasites were used for each sample and all incubations were 1 h, slowly rotating at 4°C in 200 µl of 3% BSA in PBS for blocking or antibody diluted in 3% BSA in PBS. Washes were in 200 µl cold PBS after spinning 3 min at 100xg. The parasites were blocked for 1 h and then re-suspended in 100 μg/mL rabbit anti-HRP (Sigma). After primary incubation they were washed 4 times and re-suspended in goat-anti-rabbit IgG-Alexa 488 (Invitrogen) at 1:500. After secondary incubation, the parasites were washed 3 times and transferred in the remaining 10–20 µl to a glass slide and 10 µl of mounting medium (Southern Biotech) was added and the coverslip was applied. Where noted, the parasites were imaged directly in the 96-well plate. Parasites were imaged on an Olympus microscope.

Cells were seeded on glass coverslips and incubated for 24 h. The cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS) for 25 min at 37 °C, and washed three times with PBS. The cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, washed three times with PBS, and then incubated in 5% fetal bovine serum in PBS (FBS/PBS) for 30 min at room temperature. After further PBS washes, the cells were incubated in 5% FBS/PBS with the primary antibody for 1 h, washed 3 times with PBS, and incubated in 5% FBS/PBS with the secondary antibody, DAPI, and TRITC-phalloidin. The cover slips were mounted on slides with mounting medium (Southern Biotechnology Associates, Birmingham, AL, USA), and photographed using a fluorescence microscope (AxioVert 40 CFL; Carl Zeiss AG, Oberkochen, Germany) equipped with a camera (AxioCam; Carl Zeiss AG) and powered by the AxioVision software (Carl Zeiss AG).

The cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 10 min. After washing with PBS, the cultures were blocked and permeabilized with Blocking Buffer (0.3% Tween 20, 5% skimmed milk, and 2% goat serum in PBS). Cultures were incubated overnight with primary antibodies [anti-SYN1 (1:20,000; homemade), anti-VGLUT1 (1:2000; Synaptic Systems, 135 302), anti-GAD65 (1:5000; Sigma, G5638), anti-PSD95 (1:2000; NeuroMab, 75–028), anti-Homer-1 (1:1000; Synaptic Systems, 160,011), or anti-Gephyrin (1:1000; Synaptic Systems, 147 011)] diluted in Blocking Buffer. After washing three times for 5 min in PBS, cultures were incubated for 30 min at room temperature with secondary antibodies [Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (1:200; Invitrogen, A32733) or Dylight 649, Goat Anti-Mouse IgG (1:200; Abbkine, A23610)]. Samples were placed in mounting medium (Southernbiotech, 0100–01) after washing them three times for 5 min in PBS and once in double distilled H₂O (ddH₂O).