Anti fade fluorescence mounting medium

After coating glass coverslips with Geltrex (1/100, Gibco, Waltham, MA) for 24 hours, cells were seeded and incubated overnight on the coated coverslips. The peptide cell staining was performed by removing the media and incubating the cells with PBS-1%-BSA for 15 minutes at 4°C. Biotinylated peptides (10 μM, EV-b or NT-b) were incubated for 20 minutes at 4°C, washed with PBS-1%-BSA for 5 minutes, and fluorescent-labeled streptavidin (1/100, AlexaFluor 647, Invitrogen, Waltham, MA) was added and incubated for 20 minutes at 4°C. The cells were then washed (PBS-1%-BSA, 5 minutes), fixed with 4% paraformaldehyde (20 minutes, RT), incubated with a 4’,6-diamidino-2-phenylindole (DAPI) solution (PBS-1%-BSA + 100 ng/mL DAPI, 20 minutes, RT) and mounted with Anti-Fade Fluorescence Mounting Medium (Abcam, Cambridge, UK). Z-stack images were acquired with a Leica SP8 confocal microscope at 63 magnification 1X and 3X. All images were post-processed identically with Image J software (Bethesda, MD).

Tumor cells were plated into the poly-D-lysine (Millipore sigma) overnight coated slides and incubated for an hour before adding NK cells. After co-culturing for 3 hours, cells were fixed with 4% PFA and permeabilized with 0.3% Triton X-100. Then stained with F-actin antibody conjugated with Alexa 488 (Invitrogen, A12379) for an hour and mounted with anti-fade fluorescence mounting medium (Abcam). Confocal data was generated on a Nikon AX-R Confocal Microscope (100X objective lens, oil immersion, Numerical Aperture: 1.45, Refractive Index: 1.515) which was purchased with support from the Office of Research Infrastructure Programs (ORIP), a part of the NIH Office of the Director under grant OD030233. Camera setting was set in multi-channel detector mode (FITC and mCherry), Galvano unidirectional scanner with Band scan mode, 4x line averaging, 1.0 μsec dwell time. Images were analyzed with NIS-Elements software (Nikon, ver.5.21.00) and ImageJ^{52 (link)}.

Briefly, live, calcofluor white (Sigma-Aldrich, St. Louis, USA)-stained (0.25 μg/mL for 30 min; room temperature [RT]) C. auris cells at MOIs of 1 and 5 were added for 2 h to 1 × 10⁵ neutrophils seeded onto 0.01% poly-l-lysine (Sigma-Aldrich)-treated sterilized glass coverslips. Before adding yeasts, neutrophils were serum starved (1 h), preincubated with pGSN (1 h), and washed with PBS. After 2 h of infection, the coverslips were washed with PBS and fixed in 3.7% paraformaldehyde (PFA) for 15 min at RT. After permeabilization (0.1% Triton X-100; 10 min; RT) and blocking (0.1% bovine serum albumin; 30 min; RT), the cells were washed and stained with phalloidin-Texas red (Thermo Fisher Scientific, Waltham, USA) at a dilution of 1:40 for 1 h, at RT, in the dark. The coverslips were mounted with antifade fluorescence mounting medium (Abcam, Cambridge, UK) and examined by confocal microscopy. The results are presented as the engaged cells (the percentage of cells uptaking or adherent to fungal cells) and phagocytic index (the total number of fungal cells taken up per 100 cells). Data were obtained from 5 separate experiments by analyzing at least 500 neutrophils per coverslip.

Slices with a 5 μm thickness were fixed in acetone and blocked with 1% BSA plus Fc Block (1:100) (BD Bioscience, San Diego, CA, USA) for 30 min at 25 °C. Next, the slides were stained with anti-CD4 (PE) and anti-GL-7 (FITC) (BD Biosciences, San Diego, CA, USA) or anti-CD19 (PE) (BD Biosciences, San Diego, CA, USA) and primary anti-AID antibodies (Abcam, Waltham, MA, USA) for 2 h at 25 °C. For AID, secondary anti-rabbit FITC antibodies (Abcam, Waltham, MA, USA) were added for 1 h at 25 °C. After washing, slides were mounted with Anti-Fade Fluorescence Mounting Medium (Abcam, Waltham, MA, USA). Sections were analyzed by immunofluorescence microscopy (AxioVert.A1/Zeiss, Jena, Germany).

For histological analysis, mouse ovaries were fixed in Bouin’s solution overnight, dehydrated in an ethanol gradient, embedded in paraffin, and cut into 5 μm sections. The sections were then deparaffinized, rehydrated, and stained with H&E following standard procedures. For IF experiments, WT and p63^+Δ/TID (or p63^+/R647C) ovaries or skin samples were fixed in 4% PFA, embedded in paraffin, and sectioned at 5 μm thickness. The slides underwent antigen retrieval and were then blocked, followed by incubation at 4°C overnight with the following primary antibodies: anti-DDX4 (1:600 dilution, Abcam, catalog ab27591); anti-p63 (1:200 dilution, Abcam, catalog ab124762); anti–cleaved-PARP1 (1:400 dilution, Cell Signaling Technology, catalog 94885S); anti-K10 (1:150 dilution, Abcam, catalog ab76318); anti-K14 (1:100 dilution, Santa Cruz Biotechnology, catalog sc-53253); and anti-Ki67 (1:200 dilution, Abcam, catalog ab15580). The slides were then washed in PBS, incubated with secondary antibodies (Alexa Fluor 568–conjugated donkey anti-rabbit antibody or Alexa Fluor 488–conjugated donkey anti-mouse antibody, 1:800 dilution, Invitrogen, Thermo Fisher Scientific, catalogs A21202 and A10042) for 1 hour at room temperature, and then sealed in antifade fluorescence mounting medium (Abcam). Images were captured with a fluorescence microscope (Olympus) and analyzed with ImageJ.

IHC was carried out using testicles that are shown positive or negative results in RT-PCR. The testicle tissues were fixed in formalin, embedded in paraffin, and cut into 3-μm sections. To detect PEDV, slides were deparaffinized in xylene and rehydrated through graded ethanol. Endogenous peroxidase activity in the tissue was blocked with 3% hydrogen peroxide. After blocking with 5% normal goat serum, slides were incubated with homemade anti-PEDV monoclonal antibody as 1:100 dilution in 0.1 ml of SignalStain® Antibody Diluent (Cell Signaling Technology, Beverly, MA). Then, Goat anti-mouse immunoglobulin G (IgG) Alexa Fluor Plus 488 (ThermoFisher Scientific, Waltham, MA) was applied as a secondary antibody at a concentration of 1 μg/ml for 2 h at room temperature. Hoechest 33,342 dye was used for staining nuclei, and slides were mounted in Anti-fade Fluorescence Mounting Medium (Abcam, Cambridge, MA) and then washed. The stained tissues were examined under fluorescence microscope (Optinity KI-2000F; Korea Lab Tech, Korea), and images were analyzed by using Optinity OpticView v3.7 software (Korea Lab Tech).