Superfrost plus glass slide

Adult worms were dissected in 4 µl egg buffer (25 mM HEPES, pH 7.4, 118 mM NaCl, 48 mM KCl, 0.2 mM CaCl_2, 0.2 mM MgCl₂) on a 18 mm X 18 mm coverslip. 4 µl of 4% formaldehyde (in egg buffer) were added, and the solution was mixed by tapping the coverslip before it was placed onto a Superfrost/Plus glass slide (Fisherbrand, 12-550-15). Fixed samples were incubated for 5 min at room temperature in a humid chamber, then frozen in liquid nitrogen for at least 1 min. Coverslips were removed quickly with a razor blade, and slides were placed immediately into PBS-T (PBS with 1 mM EDTA and 0.5% Triton X-100). Slides were subjected to three 10 min washes in PBS-T at room temperature. Slides were dehydrated in 95% ethanol for 10 min at room temperature followed by either the FISH or immunofluorescence protocol below.

Whole blood (23 mL) was collected in BD Vacutainer EDTA tubes (Becton Dickinsons, Germany) or CellSave tubes (Janssen Diagnostic, Raritan NJ, USA) and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient (OncoQuick, Greiner BioOne, Germany). All mononuclear cells were collected from the interphase layer, washed two times in phosphate buffer saline (PBS) and finally spun down at 150 g for 5 min at room temperature (RT) on a SuperFrost® Plus glass slide (ThermoFischer Scientific). Cytospins were dried for 12-24 h at RT and then stained or stored at − 80 °C.

For atomic force microscopy (AFM) analysis, denuded DMs were rinsed with deionized water. They were spread on top of a thin layer of 2% agarose gel and allowed to dry completely. To avoid any drift of the samples during measurements, they were firmly mounted onto a Superfrost-plus glass slide (Thermo Fisher Scientific) by slightly melting of the gel. Surface topography analysis was performed by static force mode with an Easyscan 2-controlled FlexAFM (Nanosurf, Liestal, Switzerland) equipped with a contact mode cantilever (ContAI-G, Budget Sensors, Sofia, Bulgaria) with a tip radius of 10 nm and nominal spring constant of 0.2 N/m. The nanostructures of enzymatic-treated DM and intact corneal endothelium were characterized from four randomly regions within each sample, with 256 x two-direction lines scanned at 2 μm/s. Surface roughness was calculated with Mountains SPIP 9 software (Image Metrology A/S, Copenhagen, Denmark) by means of root mean square (Sq) values of the surfaces within selected areas.

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.