Eclipse ni e

Brains were post-fixed in 4% PFA for 24 h at 4 ºC and transferred into 30% sucrose solution at 4 ºC. Brains were frozen, sectioned at 30 µm and every 6^th coronal slice (spanning the whole brain) was immunostained for rat MHC class II with anti-rat RT1B (clone OX-6; BD Biosciences, Mississauga, ON, Canada) to detect microglial activation^{30 (link)}. Specifically, free-floating sections were treated with 1% H₂O₂ and blocked with horse serum (Vector Laboratories, Inc., Burlingame, CA, USA). The sections were incubated with anti-rat RT1B in blocking serum overnight at 4 °C, followed by 1-h incubation with biotinylated 2° antibody (BA-2000; Vector Laboratories, Inc.) in serum at room temperature. Sections were then incubated with avidin–biotin complex (Vector Laboratories, Inc.) for 1 h followed by 0.05% diaminobenzidine. After washing, sections were mounted on slides with 0.3% gelatin and air-dried, followed by dehydration, clearing in xylenes, and mounting with DePex mounting medium (Electron Microscopy Science, Hatfield, PA, USA). After examination under high magnification, stitched images of brain sections were acquired using an upright microscope (Nikon Eclipse Ni-E, Nikon DS Fi2 color camera, NIS Elements Imaging, Mississauga, ON, Canada).

A combination of differential interference contrast (DIC) microscopy (Model: Leica DMR microscope, Leica Microsystems (Schweiz) AF- Heerbrugg, Switzerland; Camera: Jenoptik Gryphax Kapella, Jenoptik, Jena, Germany) and bright-field microscopy (Model: Nikon Eclipse Ni-E, Nikon Corporation, Tokyo, Japan) was used to visualize and characterize the plastid development between three species as the fruit developed. Free hand sections of fresh fruit flesh were cut using razor blades and mounted on glass slides without staining.

To visualize binding and phagocytosis of bacteria by neutrophils, neutrophils (isolated from separate donors) were diluted to 2.0 × 10⁶ cells/ml and seeded onto tissue culture-treated glass coverslips with 1% HSA or 1% NHS (45 (link), 46 (link)). Bacteria were resuspended to an OD₆₀₀ of ∼0.8 in PBS, stained with 5 μg/ml CFSE (Tonbo Biosciences, San Diego, CA) for 20 min at 37°C, and washed twice with PBS. CFSE-stained bacteria were added at an MOI of 10:1 and centrifuged at 1,000 rpm for 2 min. Samples were incubated for 30 min at 37°C with 5% CO₂. Cells were washed twice with PBS to remove nonadherent bacteria and were fixed at RT for 15 min using 4% paraformaldehyde in PBS. Samples were incubated with anti-K. kingae antibody (1:500) overnight at 4°C. The anti-K. kingae antibody was generated against an acetone powder of K. kingae strain KK01 whole bacteria. Cells were washed twice with PBS and incubated with a 1:500 dilution of goat anti-guinea pig IgG DyLight 649-conjugated antibody (Rockland, Limerick, PA).
Visualization of bacteria-neutrophil association was performed by microscopy using a Nikon Eclipse Ni-E (Nikon Instruments, Inc., Melville, NY) with either a 20× objective or 60× oil objective. For quantification, 100 neutrophils per replicate were chosen at random for analysis; the cell-associated and extracellular bacteria were counted by a blinded reader.

For the assessment of cell concentration, a Burker counting chamber was used. The depth of the counting chamber was 0.1 mm, and the counting area was 0.04 mm². Cells were counted daily under the microscope (Nikon Eclipse Ni-E, Nikon Corporation, Tokyo, Japan). The cell concentration was calculated at a 250 × 1000 × dilution rate (cell number/mL).