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Anal Biochem

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Inhale slowly through your nose, hold for a few seconds, then exhale through your mouth

Immunofluorescence staining of SIOs SIOs were fixed in 4% paraformaldehyde (Biosharp, Hefei, China) at room temperature for 20 min, washed three times with PBS, blocked with blocking buffer at room temperature for 1 h, incubated overnight with a primary antibody at 4 C, washed three times with PBS, incubated with a secondary antibody at room temperature for 90 min, washed three times with PBS, stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich Corporation) for 10 min, washed three times with ultrapure water, mounted on glass slides, and observed with a laser confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany)