Lsm pascal confocal laser scanning microscope

For microscopic examination, the hepatic macrophages were washed three times with cold PBS, fixed with 2% paraformaldehyde in PBS for 30 min, then permeabilized with 0.2% (w/v) Triton X-100 in PBS for 5 min. Hepatic macrophages were then blocked with 0.5% bovine serum albumin in PBS for 1 h before being incubated with an Alexa-Fluor 488 Avidin-labeled antibody detecting F4/80, a macrophage marker (Invitrogen, Carlsbad, CA, USA). Nuclei were visualized by staining with 4',6-diamidino-2-phenylindole (DAPI), and cells were observed under an LSM PASCAL confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

To gain insight into the location and expression level of regnase-1 (MCPIP) in KCs in the liver under different milieu, double immunofluorescence was performed with a combination of diluted antirabbit F4/80 and monoclonal antibody against MCPIP. Fresh frozen liver tissue sections on coverslips from all groups were sequentially reacted with F4/80 and anti-MCPIP for staining as the primary antibody overnight, followed by reaction with horseradish peroxidase-conjugated secondary antibody (Histofine simple stain MAX PO; Nichirei Inc., Tokyo, Japan) as the second antibody. The nuclei were finally counterstained by 4′,6-diamidino-2-phenylindole (DAPI) for 3 min for cell localization. Fluorescence images were captured under an LSM PASCAL confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany), with excitation spectral laser lines at 405 and 594 nm.

Phalloidin staining was performed on 96 hpf embryos (N = 8) as described³⁵ . Following fixation in 4% PFA and permeabilization with 50 µg/ml Proteinase K, embryos were incubated in Alexa-Fluor 488 conjugated Phalloidin (Invitrogen, California, USA, Cat. No. A12379) overnight at + 4 °C. Embryos were then mounted onto glass bottom dishes and photographed with LSM Pascal laser scanning confocal microscope (Carl Zeiss, Jena, Germany). Somite lengths were measured by using ImageJ (Version 1.49u).