Gelvatol

For immunohistochemistry, left eyes (n ≥ 3 per assay) were fixed in 4% paraformaldehyde in PBS for 15 minutes on ice, incubated overnight in PBS containing 15% and 30% sucrose and embedded in optimal cutting temperature (OCT) compound. 10 μm frozen sections were air-dried at RT and immunohistochemistry was realized as in^{10 (link)}. Nuclei were stained with 2 ug/ml Hoechst 33342 (Thermo-scientific #62249) and slides were mounted in Gelvatol pH = 8.5 (Sigma-Aldrich). For TUNEL assays frozen retinal sections (10 um) were incubated 1 h in blocking solution, and TUNEL assays was done following standard procedures from the manufacturer (In Situ Cell Death Detection Kit, Fluorescein, Roche #11684795910).

ELISA IgA anti-TTG seroreactive samples (11 donors, Table 1) were tested for IgA anti-EMA using the AESKUSLIDES-EMA kit (Ref. 512.050, Lot. no. 18044; AESKU Diagnostics, Wendelsheim, Germany). The slides were placed in a humid chamber for 30 min. The donor serum was diluted 1 : 5, placed in the well, and incubated for 30 min along with the positive and negative controls. After washing the slides, 50 μL of IgA conjugated with the FITC fluorochrome was added to each well, and the samples were then incubated for 30 min in the dark followed by washing with 1x PBS. Evans blue dye diluted at 1 : 3000 was added to each well (Sigma-Aldrich, St. Louis, MI, USA), the slides were washed and allowed to dry, and then mounting medium (Gelvatol; Sigma-Aldrich, St. Louis, MO, USA) was added to each slide. The slides were analyzed under a fluorescence microscope by two independent readers (Zeiss, Oberkochen, Germany).

Human melanoma OCT sections (5 μm) were fixed with 2% paraformaldehyde for 20 min. Sections were stained overnight with 2 µg/ml of Alexa Fluor 546 mouse anti-human TNFR2 (Santa Cruz; Ab clone D-2) and Alexa Fluor 647 rabbit anti- human SOX10 (Abcam; Ab clone SP267) antibodies. Nuclei were stained with Hoechst (Sigma) 1 mg/100 ml dH₂0 for 1 min, washed in PBS, and mounted in Gelvatol (Sigma). Large area scan images at 40 × magnification were obtained on Nikon A1 confocal microscope with NIS Elements v5.2 (Nikon Instruments Inc; Melville, NY). To confirm forced TNFR2 expression on the SK-Mel-28 cell line, 3 × 10⁵ melanoma cells were seeded in 6-well plates (Corning) and cultured overnight at 37 °C. Cells were directly fixed in the wells using 2% paraformaldehyde for 30 min at 4 °C. Cells were stained for 1 h at room temperature with 10 µg/ml of anti-human TNFR2 antibody (clone #22221; R&D Systems) and were subsequently stained with Alexa Fluor 488–conjugated goat anti–mouse immunoglobulin F(ab′)2 (Cell Signaling Technology; Danvers, MA). Nuclei were labeled with 300 nM DAPI (ThermoFisher) for 10 min at room temperature. Images at 20X magnification were generated using the BioTek Lionheart FX automated microscope and Gen5 Image + v3.05 software (BioTek).

Cells were fixed in 1.5% formaldehyde in PBS, permeabilized with acetone at −20°C for 7 min, and washed three times in PBS and once in distilled water. Water-resuspended cells were used to prepare smears, which were air-dried at room temperature and rinsed with PBS. The smears were then incubated with primary antibody (anti-FAK 1:100, anti-vinculin 1:100, anti-paxillin 1:100, anti-talin 1:100, anti-integrin-β 1:100, or anti-α-actinin 1:100) in blocking solution (1% bovine serum albumin in PBS) for 12 h at 4°C. After extensive washes with PBS, the cells were incubated with the appropriate TRITC- or Cy5-labeled secondary antibodies for 1 h at 37°C. In all cases, smears were extensively washed with PBS and mounted on cover glass slides using gelvatol (Sigma Chemical Co., St. Louis, MO) (Harlow and Lane, 2006 (link)). To confirm the specificity of the antibodies and the location of the focal adhesion proteins, three types of controls were assayed: (1) spermatozoa were only incubated with the secondary antibody, (2) spermatozoa were incubated with a non-specific primary antibody, and (3) the primary antibodies used were pre-incubated with their respective blocking peptides before the immunofluorescence assay. Spermatozoa stained under these conditions showed very faint patterns of fluorescence (Fig. S3).