Plan apochromat 63 1.4 na objective

Flash frozen 6 mm biopsy punches were thawed/fixed in ice-cold Carnoy’s buffer (60% MeOH, 30% acetic acid, 10% acetic acid) for 3 h on ice before washing as follows: 2× MeOH washes, 30 min each, 2× ETOH washes, 20 min each, 2× PBS washes, 20 min each. Tissues were then embedded and frozen in OCT media and 5 μm sections were cut and placed on slides for staining. Tissue sections were blocked with 2% donkey serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween-20 in PBS, following by staining with the following primary antibodies: mouse anti-cytokeratin clone C-51 (Thermo Fisher, 1:100), rabbit anti-mCherry polyclonal (Thermo Fisher, 1:100), and goat anti-Salmonella CSA-1 (1:100) conjugated to AlexaFluor 488 (Thermo Fisher) per manufacturer’s instructions. Secondary antibodies (1:500) were AlexaFluor 568-conjugated donkey anti-rabbit (Thermo Fisher) and CF^®633 conjugated donkey anti-mouse (Biotium). After staining, slides were stained for 10 min with wheat germ agglutinin (WGA) conjugated to CF^®405 (Biotium, 1:500), followed by fixation with 2.5% PFA for 10 min. Samples were mounted with ProLong Gold with DAPI (Thermo Fisher). Images were captured using a Zeiss LSM 710 confocal laser-scanning microscope with either a Plan APOCHROMAT ×63/1.4 N.A. objective or a ×20/0.8 N.A. objective.

RBL cells were seeded at 2 × 10⁵ cells/chamber in eight-well chamber borosilicate coverglass systems (Thermo Fisher Scientific Waltham, MA). Cells were loaded with 1 mg/ml of FITC-dextran for 48 h. Images were acquired by a Leica Sp5 laser scanning confocal microscope, equipped with a heated chamber (37 °C) and CO₂ controller (4.8%) and a C-Apochromat 363/1.2 W Corr objective or using a Zeiss LSM Pascal confocal microscope using a Plan-apochromat 63° – NA 1.4 objective (Carl Zeiss MicroImaging) or Zeiss LSM 800 microscope. Image capture was performed using the standard time-series option (Carl Zeiss MicroImaging). Images and videos were generated and analyzed using the Zeiss LSM software and ImageJ software (W. Rasband, National Institutes of Health, Bethesda, MD). Long time-lapse image sequences were captured using the autofocusing function.