Fluormount

RKO pBAR-Renilla cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. Samples were then blocked for 1 h with 5% normal goat serum (Gibco). A rabbit anti-β-catenin (1:200) primary antibody was incubated overnight. Specific secondary antibody Alexa 546 (Alexa Fluor ®546 Goat Anti-Rabbit IgG; Invitrogen) (1:2000) were incubated for 1 h at room temperature. After PBS washes, DAPI staining (4,6-diamidino-2-phenylindole) (Sigma) was performed for 10 min, and then slides were mounted with Fluormount (Sigma) and observed at confocal microscope (Leica TCS SP5).

For immunofluorescence analysis, mice were perfused with 4% paraformaldehyde (PFA) in PBS 1X after 1 mL intraperitoneal injection of 2% w/v Tribromoethanol. The colon was collected and flushed of fecal contents, post-fixed o/n in 4% PFA/PBS at 4 °C and stored in maintenance solution (30% sucrose, 0.1% NaN₂ in PBS) at 4 °C. A segment of 2 cm of distal colon was embedded in Tissue-Tek® OCT (Sakura, The Netherlands), cut at the cryostat in serial 12 μm sections and mounted on SuperFrost Plus glass slide (Thermofisher). Slides were then washed with PBS and let dry for ~ 3 h at 37 °C. Sections were then incubated with blocking solution [3.5% fat dry milk, 0.3% Triton X-100 (Tx-100), 6% normal goat serum (NGS) in PBS] for 1 h at RT and then incubated with primary antibody o/n at RT in blocking buffer. The following antibodies were used: pser129-αS and LB509 (Abcam), β-3-Tubulin and Syn204 (Cell Signaling Technology, Danvers, MA, USA), ChAT and Tyrosine Hydroxylase (TH) (Millipore, Burlington, MA, USA). On the next day, the sections were washed twice in PBS and incubated with Alexa Fluor secondary antibodies (ThermoFisher) in PBS containing 1.5% NGS, 0.3% Tx-100 for 1 h at RT. Sections were counterstained with Dapi and mounted on a glass slide using Fluormount (Sigma-Aldrich).

Frozen sections of the distal colon were embedded in Tissue-Tek^® OCT (Sakura, The Netherlands), cut at the cryostat in serial 12 μm sections and mounted on Super-Frost Plus glass slide (Thermo fisher). For immunostaining slides were incubated with blocking solution [3.5% fat dry milk, 0.3% Triton X-100 (Tx-100), 6% normal goat serum (NGS) in PBS] for 1 h at RT and then incubated with TLR-2 (Santa Cruz, Dallas, TX, USA) and GFAP (ab7260, Abcam) antibody o/n at RT in blocking buffer. Slides were washed twice in PBS and incubated with Alexa Fluor secondary antibodies (Thermo Fisher) in PBS containing 1.5% NGS, 0.3% Tx-100 for 1 h at RT. Sections were counterstained with Dapi and mounted on a glass slide using Fluormount (Sigma-Aldrich). Image acquisition was carried out using a Zeiss Apotome fluorescent microscope, using a 20× objective.

Three groups of ten l-L3 instar larvae treated with PF-04449913 or DMSO and expressing GFP in all hemocytes under the control of the hml-Gal4 driver, were bled into 30 μl of Drosophila Ringer solution, labeled with Cy3 conjugated phalloidin (20 μg/ml, Sigma) that binds filamentous actin, and mounted under a coverslip with Fluormount. In order to evaluate the number of circulating hemocytes, GFP expressing cells were counted in three random fields per coverslip, and the average number of hemocytes per field between the coverslips was calculated. To evaluate the number of lamellocytes, we counted the phalloidin/GFP positive cells that showed big size and flat shape (compared to the small size and round shape of plasmatocyte and crystal cells) present under each coverslip and the average between samples was calculated.