Lsm 880 microscope

For migration imaging, IgA-Cre/YC3.60^flox mice (8–12 weeks-old) were anesthetized with a mixture of three types of anesthetic agents as described previously (19 (link)). An incision was carefully made in the abdominal wall, and the small intestine was exposed. PPs were identified by naked eyes. About 8000 sorted CD11b⁺IgA⁺ PP B cells and 30 000 CD11b⁻IgA⁺ PP B cells were labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then injected to a PP of an IgA-Cre/YC3.60^flox transgenic mouse directly by a 25 μl syringe (Trajan Scientific and Medical, Australia). The PP with transferred cells was observed under an LSM 880 microscope (Carl Zeiss, Germany). Images were analyzed with ZEN2009 software (Carl Zeiss, Germany). After one-hour observation of the PP under a microscope, the abdominal incision was carefully closed with an ELP Skin Stapler (Akiyama Co. Ltd. Japan). Forty hours after transfer, under anesthesia, the PP with transferred cells was observed again to identify the localization of transferred CD11b⁺IgA⁺ PP B cells under an LSM 880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).

For confocal imaging of conical cells from the side, petal blades were folded back, allowing for the side view of conical cells, and stained with a solution containing 10 μg/ml propidium iodide for more than 10 min. Petal samples were imaged with a Zeiss LSM 880 confocal laser scanning microscope (excitation at 514 nm, emission 550-700nm). For live-confocal imaging of cortical microtubules, non-folded petals stably expressing GFP-TUA6 were imaged with a Zeiss LSM 880 confocal laser scanning microscope (excitation at 488, emission 500-570nm). Serial optical sections were taken at 0.5-μm increments with a 40 × water or 63 × oil lens, and then were projected on a plane (i.e. maximum intensity) using Zeiss LSM 880 software.
For CM-H2DCFDA staining, petal samples were incubated in 50 mM phosphatic buffer solution (PBS, pH 7.4) containing 10 μM CM-H2DCFDA (Invitrogen, C6827) for 30 min, and then the samples were washed for three times with PBS, and observed with the Zeiss LSM 880 microscope (excitation 488 nm, emission 500–570 nm) or the Zeiss observer A1 inverted microscope. For Dihydroethidium (DHE) staining, petal samples were incubated into 50 mM PBS (pH 7.4) buffer solution containing 40 μM DHE (Sigma, D7008) for 30min, and then visualized with the Zeiss LSM 880 microscope (excitation 514, emission 520–600 nm) or the Zeiss observer A1 inverted microscope.

Free-floating 30 μm of the coronal sections of brain were stained with anti-Iba-1 overnight at 4°C, and secondary antibody was incubated for 2 h at RT. Nuclei were stained with DAPI. Confocal images were taken at ×40 oil immersion objective with 1-μm interval for 20 μm depth using LSM880 Zeiss microscope. Microglia structure was rendered using IMARIS software.

Images of the human bone slices were taken using LSM 880 Zeiss Microscope (Zeiss, Oberkochen, Germany) with an LD LCI plan-apochromat 25×/0.8 l mm korr DIC M27 objective (Zeiss, Oberkochen, Germany). Two lasers (561 nm at 10–22% power and 405 nm at 1.1–1.8% power) were used and images were taken as a Z-stack. A spectral scan was run and collected on a control bone that was stained with 1 µM of Calcein red-orange AM (Calcein acetoxymethyl ester). This was to establish the spectral peak of the dye (590 nm emission spectrum) and was then set as the control peak. Linear mixing was used, in order to identify the defined spectrum and remove autofluorescence of the stain (in some cases fingerprinting technology was also used).