Fluoromount

Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33,342, Fluoromount, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Rhodamine-123 and Fetal Bovine Serum (FBS) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were procured from Hi-media Pvt. Limited, Mumbai (India). Rabbit monoclonal Bcl-xl, p-53, p-NF-κB, Caspase3, and Caspase9 antibodies and anti-rabbit- HRP secondary antibody were obtained from Cell Signaling Technology, Danvers, MA, USA. PVDF membrane (MDI, Ambala). The RT-PCR chemical kit was purchased from Bio-Rad, CA, USA. The BD Cycletest plus DNA Kit was from BD Biosciences, San Jose, CA, USA. Chemicals and reagents of analytical (AR) grade were used to perform the experiments.

Immunostaining was performed as described earlier [30 (link), 31 (link)]. Briefly, coverslips containing 200–300 cells/mm² were fixed with 4 % paraformaldehyde for 15 min, followed by treatment with cold methanol (−20 °C) for 5 min and two rinses in PBS. Samples were blocked with 2 % BSA in PBS containing Tween 20 (PBST) for 30 min followed by incubation in PBST containing 1 % BSA and mouse anti-CD68 (1:200), goat anti-IBA1 (1:200), rabbit anti-iNOS (1:200), mouse anti-GFAP (1:500), mouse anti-MBP (1:200), and goat anti-MAP-2 (1:100). After three washes in PBST (15 min each), slides were further incubated with Cy2, Cy5, and DAPI (Jackson ImmunoResearch, West Grove, PA, USA). After secondary antibody incubation, coverslips were rinsed in 1X PBS, mounted on slides in Fluoromount (Sigma) and imaged using an Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.

J774 macrophages grown in low glucose DMEM (D5546, Sigma) with 10% FBS were plated at cell density of 1 × 10⁵ cells per well onto sterile 18 mm glass microscopy cover slips (VWR) in a 24-well plate and incubated at 37°C with 5% CO₂ overnight. Macrophages were activated by adding 1 ml of serum-free DMEM medium containing 15 μl of a 10 μg/ml phorbol 12-myristate 13-acetate (PMA) stock (P8139, Sigma). Macrophages were incubated for 1 h at 37°C and 5% CO₂. Immediately medium was removed and cells were washed twice in PBS. Overnight cultures of C. albicans containing the fluorescent metal sensors were washed in PBS and adjusted to a cell density of either 2 × 10⁶ or 5 × 10⁶ cells/ml. Nine hundred and fifty microliter of fresh serum-free DMEM with 50 μl of the Candida suspension was added to the macrophages and incubated for a further hour at 37°C and 5% CO₂. After the incubation the medium was removed, cells were washed twice with PBS followed by fixation in 4% paraformaldehyde for 10 min. The fixative was removed, cells were washed twice in PBS and mounted onto microscopy slides with Fluoromount (F4680, Sigma). Images were taken at 40 × magnification using a Zeiss Axio observer microscope using DIC, GFP, and Texas Red channel. Images were acquired through Axio Zen software (Zeiss).

Cells were fixed with 4% paraformaldehyde for 30 min before embedding in OCT compound (Sakura, Tokyo, Japan) and subsequent snap-freezing. Tissue samples were fixed with 4% paraformaldehyde for 48 h, processed for paraffin embedding, and deparaffinised before preblocking. Immunofluorescence staining was carried out on serial sections of 5 μm thickness. Samples were preblocked with phosphate-buffered saline (PBS) containing 2% donkey serum, 2% bovine serum albumin (BSA), and 0.2% NP-40 for 30 min at room temperature. Primary and secondary antibodies were diluted with PBS containing 2% donkey serum, 2% BSA, and 0.1% NP-40. Primary antibodies were applied overnight at 4 °C and are summarised in Supplementary Table 2. Following three washes with PBS, secondary antibodies were applied to visualise primary antibodies for 60 min at room temperature (protected from light). Fluorescence labelling was performed using Alexa Fluor 488, 594, and 647 secondary antibodies (Thermo Fisher Scientific, all diluted 1:300). Following three washes with PBS, the slides were mounted with Fluoromount (Sigma–Aldrich). Images were acquired using a laser confocal microscope (Olympus, Tokyo, Japan).

Cells were washed with phosphate buffered saline (PBS) and fixed for 30 min at room temperature with 4% paraformaldehyde (containing 4% sucrose; Sigma). Cells were permeabilized in 0.5% NP40 in PBS for 5 min at room temperature. All primary and secondary antibodies, dilutions and suppliers can be found in Table 1. Primary antibodies were incubated overnight at 4 °C. Corresponding Alexafluor secondary antibodies (1:500, Thermo Scientific) were incubated for 1 h at room temperature before mounting with Fluoromount (Sigma).

Cells were plated at a density of 20,000 - 35,000 cells per 10 mm² coverslip and left to adhere overnight before fixing with 4% paraformaldehyde. Fixed cells were permeabilized with 0.3% Triton X-100, washed in PBS, and treated with blocking solution (10% goat serum, 2% BSA, 5% sucrose in PBS) for 45 min. Cells were incubated overnight at 4°C with primary antibodies in blocking solution and then used in in situ proximity ligation assays (PLAs; Duolink kits, Sigma). Briefly, cells were treated for 1 h at 37°C with plus and minus probes directed at rabbit and mouse antibodies, respectively. Then probes were ligated for 30 min at 37°C. Next, in situ PCR amplification was done with Alexa 568- or Alexa 488-conjugated oligonucleotides for 2.5 h at 37°C. Samples were stained with Hoechst 33452 (Thermo Fisher Scientific) and Alexa 488- or Alexa 568-conjugated phalloidin (Thermo Fisher Scientific), mounted on microscope slides using Fluoromount (Sigma), and imaged under a confocal microscope (Nikon Ti-Eclipse or Leica SP8) with a 60X objective.