Axiovert a 1 fluorescent microscope

DiO (Thermo Fisher Scientific) was dissolved in DMSO (Sigma) at 2 mg/ml and stocked at −30 °C. Well-fed worms were collected from a 60-mm plate with 1 ml M9 buffer and pelleted at 1400 g for 2 min. Worms were resuspended in 1 ml M9 buffer and DiO was added (final 0.01 mg/ml). The mixture was slowly shaked for 3 hours. Worms were washed with M9 buffer twice. Fluorescent signal was observed with an Axiovert A1 fluorescent microscope (Carl Zeiss).

PC12 cells were plated in 96-well plates, serum-deprived for 4 h and subsequently treated with or without NGF (100 ng/mL) or ENT-A010 (500 nM) in the presence or absence of GW441756 TRKA inhibitor (20 μM, G-190, Alomone Labs, Jerusalem, Israel) for another 24 h. The CellTox assay (Promega, Leiden, Belgium) was performed according to the manufacturer’s instructions. Hoechst (1:10,000, Invitrogen, MA, USA) was added for 30 min along with CellTox reagent and cells were imaged with a Zeiss AXIO Vert A1 fluorescent microscope. The number of CellTox⁺ (dead) cells was normalized to the number of Hoechst⁺ cells, the latter depicting the total number of cells, per image. The numbers of CellTox⁺ and Hoechst⁺ cells were determined using the FIJI software.

HEK293 cells were seeded on coverslips coated with poly-l-lysine. Cells were cultured for 30 h post-transfection and then fixed with methanol. Nuclei were stained with Hoechst 33342. Fluorescence images were acquired using an Axiovert A-1 fluorescent microscope with ZEN Image software (Zeiss).

CellTox assay (G8742, Promega, Leiden, Belgium) was used to assess the survival of PC12 cells under serum deprivation conditions. PC12 cells were plated in 96-well plates, starved from serum for 4 h, and subsequently treated with NGF (100 ng/mL) or compound (500 nM) for 24 h. CellTox assay reagents and Hoescht (1:10,000, H3570, Invitrogen, Massachusetts, USA) were then added to each well for 30 min, and then, cells were imaged with a Zeiss AXIO Vert A1 fluorescent microscope. CellTox positive cells were normalized to the total number of cells for each image.

CellTox assay (G8742, Promega Corporation, Maddison, WI, USA) was used to assess survival of PC12 cells under serum deprivation conditions. PC12 cells were plated in 96-well plates, starved of serum for 4 h and subsequently treated with NGF (100 ng/mL) or ENT-A013 (500 nM) in the presence or absence of TrkA inhibitor GW441756 (20 μM, G-190, Alomone labs, Jerusalem, Israel) for 24 h. CellTox assay reagents and Hoescht (1:10,000, H3570, Invitrogen, Waltham, MA, USA) were added to each well for 30′ and cells were then imaged with a Zeiss AXIO Vert A1 fluorescent microscope (Zeiss, Jena, Germany). CellTox positive cells were normalized to total number of cells for each image.

Cells were grown on coverslips coated with poly-L-lysine (Sigma-Aldrich). 48 h post-transfection, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences). Nuclei were stained with Hoescht 33342 (Invitrogen). Fluorescence images were acquired with an Axiovert A-1 fluorescent microscope and ZEN Image Software (Zeiss). For quantitative microscopy, two (for YFP-TBR1 nuclear aggregation) or four (for CASK and mCherry-TBR1 nuclear aggregation when co-expressed with YFP-TBR1 variants) 3 × 4 stitched images were taken with a 40x objective for each experiment and manually counted using ImageJ software. Quantification was conducted with the experimenter blinded to the conditions.

A BrdU incorporation assay was used in order to determine the effect of HES and H. polygyrus antigen on CT26.WT DNA synthesis and cell division, and 2 × 10⁵ cells were seeded on a sterile glass coverslip in a 35 mm dish, treated with HES (10 µg), H. polygyrus antigen (10 µg), or 1X PBS, and allowed to adhere overnight. A total of 10 µm BrdU (Sigma-Aldrich, Saint Louis, USA) was added to the medium for 8 h, followed by fixation with Carnoy’s fixative for 20 min at −20 °C. Cells were incubated in 2 M HCl for 1 h at 37 °C, neutralized in 0.1 M pH 8.5 borate buffer, and washed with PBS/0.5% Tween20. Cells were then incubated for 30 min at 37 °C in blocking buffer (5% sheep serum in PBS/0.5% Tween20). BrdU was detected with a mouse monoclonal anti-BrdU (clone: BMC9318, Roche, Mannheim, Germany) for 30 min at 37 °C, followed by a secondary IgG coupled to Alexa488 (RRID: AB_2534069, Thermo Fisher Scientific, Wilmington, USA) for 30 min at 37 °C. Cells were washed with PBS/0.5% Tween20, incubated in 1 µg 4’6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Inc, Eugene, USA) for 10 min at room temperature, in the dark, and washed with PBS/0.5% Tween20. The coverslips were then mounted onto microscope slides, stored in the dark in a humidifying chamber overnight at 4 °C, and visualized by fluorescence microscopy using an Axio Vert.A1 Fluorescent microscope (Zeiss, Jena, Germany).