Cytospin

MIS/AMHRII expression was immunocytochemically detected using the Invitrogen Histostain Plus AEC kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cultured human endometrial cancer cells were harvested, 200 µl of a 1×10⁵ cells/ml suspension was centrifuged with a cytospin (Thermo Electron Corp., Cheshire, WA7, UK) at 1,000 rpm for 5 min, and the cell pellet was transferred to ProbeOn Plus slides (Fisher Scientific). These slides were then treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase activity and washed three times with PBS. After treatment with donkey serum (Invitrogen) for 30 min to block non-specific protein binding, the slides were incubated with rabbit polyclonal anti-human MIS/AMHRII antiserum (153P) at 4°C overnight. The slides were rinsed in PBS three times and incubated with biotinylated anti-rabbit IgG (Invitrogen) for 30 min. After another three PBS rinses, the streptavidin HRP detection system (Invitrogen) was applied to the slides for 30 min to induce the biotin-avidin binding reaction. The slides were treated with AEC for 10 min at room temperature, counterstained with hematoxylin, and mounted with glycerol gel. Three hundred endometrial cancer cells were counted in five different microscopic fields, and the mean percentage of immunopositive cells was calculated.

Six micrometers cryosections were obtained from skin biopsies and prepared on poly-L-lysine coated glass slides (Cytospin, Thermo Scientific). Staining for telomere-associated ɣH2AX foci (TAF) was then performed. Sections were stained for ɣH2AX (Ser139, Cell Signaling #9718, 1:250), p16^INK4a (Abcam, ab108349, 1:100 or Sigma SAB5300499, 1:100) and HLA-E (clone 3D12, eBioscience, 1:100), followed by incubation with secondary antibody conjugated to various fluorochromes and washed in formamide/SSC prior to mounting with Vectorshield/DAPI (Vector Laboratories). Slides were air dried prior to hybridisation for 2 h with 40 pM PNA probe targeting the TelC telomeric repeat (Panagene, TelC Cy3, #14 1224PL-01). Imaging of TAF foci was then performed using a Leica SPE2 confocal microscope (Leica Microsystem). Imaging consisted of obtaining Z-stacks with a step-size of 0.5 µm. Analysis was performed using Fiji image analysis software (Fiji.sc).

Bacterial binding to CLR-hFcs was visualized by staining bacteria with the indicated fusion proteins at 200 ng/mL in lectin-binding buffer for 2 h at 4°C, followed by incubation with an anti-human IgG (Fc)-PE or Alexa Fluor 488-labeled antibody (Jackson ImmunoResearch Labs) diluted 1:5,000. When indicated, bacteria were also stained with Salmonella Vi antiserum (Bio-Rad) at 1:5,000 and an anti-rabbit Alexa Fluor 594 at 1:5,000. When GFP-expressing bacteria were not used, bacteria were visualized with Hoechst 33342 staining (1:600). Stained samples were mounted onto slides using a Cytospin (Thermo Fisher Scientific) for 10 min at 22 × g. Samples were then mounted with coverslips using proLong antifade mountant (Invitrogen). Binding was visualized by fluorescence microscopy (Zeiss Axio Observer).

Cells were grown on coverslips, and mitotic cells were arrested with nocodazole for 1–6 h. After medium removal, hypotonic buffer (0.8% [wt/vol] sodium citrate/H₂O; freshly added: 10 mM NaF; 0.5 mM Na₃VO₄; 2 µg/ml aprotinin, 1 µg/ml leupeptin; and 1 mM PMSF) was added drop-wise, and cells were swollen for 15 min at room temperature. Cells were centrifuged on coverslips with a CytoSpin (Thermo Fisher) at 250 g for 5 min and processed for immunofluorescence staining.