Alexa fluor 647

Oocytes were fixed in 4% paraformaldehyde for 30 min, washed 3 times with 0.1% Tween-20 and transferred to 0.1% Triton X-100 overnight for permeabilization. Permeabilized oocytes were washed 3 times and then blocked with 1% BSA for 1 h.
Blocked oocytes were successively incubated with the primary and secondary antibodies Rabbit anti-Parkin antibody (ab233434, Abcam, Boston, MA, USA) and Goat anti-rabbit IgG H&L (Alexa Fluor^® 647) (ab150079, Abcam, USA), washed with PBS 3 times and the fluorescence aggregation of Parkin was observed with LSCM.
For colocalization of LC3B and TOMM20, blocked oocytes were incubated with Rabbit Anti-LC3B (ab192890, Abcam, USA) and Mouse Anti-TOMM20 (ab283317, Abcam, USA) for 1 h, then incubated with secondary antibodies Goat anti-mouse IgG H&L (Alexa Fluor^® 488) (ab150113, Abcam, USA) and Goat anti-rabbit IgG H&L (Alexa Fluor^® 647) (ab150079, Abcam, USA), washed with PBS 3 times and observed with LSCM. The overlapping coefficient was analyzed using Image J as the fraction of VDAC1 overlapping LC3B.

The xenograft mice were sacrificed, and the tumors were freshly collected to perform the experiment. Then, formalin-fixed and paraffin-embedded sections of 5 µm thickness were dried at 60 °C for 30 min, deparaffinized in xylene, rehydrated through graded alcohols, and immersed for 15 min in PBS buffer. For antigen retrieval, the sections were microwaved in 0.01 M citrate buffer (pH 6.0) for 20 min. After that, the endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 min. The sections were incubated for 60 min with 5% BSA and 0.3% Triton X-100 in PBS-T to block nonspecific staining and then incubated with indicated primary antibodies overnight. Alexa Fluor^® 555 (Abcam, Cat#ab150074, UK), Alexa Fluor^® 647 (Abcam, Cat#ab150115, UK), and Alexa Fluor^® 647 (Abcam, Cat#ab150131, UK) were diluted in IF-blocking buffer. The specimens were washed with PBS, and then the nuclei were stained with DAPI (Invitrogen, Cat#P36931, USA) for 20 min. The slides were mounted with VECTASHIELD Antifade Mounting Medium for Fluorescence (Vector Laboratories, Cat#H-1000, Burlingame, CA, USA). The samples were imaged using a laser-scanning confocal microscope (Nikon A1), and fluorescence was analyzed. Three to four images were randomly taken per slide for analysis.

Mice were anesthetized and perfused with 4% paraformaldehyde in a 0.1 m PBS buffer at ZT8 (4 PM). The whole brain was kept in 4% paraformaldehyde at 4 °C for several days, after which some slice sections with 150 µm thickness that contained the SCN were cut in the coronal section using a micro‐slicer (DTK‐1000N; Dosaka).
Subsequently, immunofluorescence microscopy was conducted to detect Per1 expression using GFP reporter signals (in Per1::GFP Tg brain slice), after which intact neurons were immunostained using a 1 : 500 dilution of anti‐rat NeuN (ab279297; Abcam, Biomedical Campus, Cambridge, UK) primary antibody, followed by a donkey anti‐rat IgG (Alexa Fluor^® 647; ab279297; Abcam) secondary antibody at a 1 : 500 dilution. Afterward, nuclei were stained with DAPI (DOTTIE LD034, DOJINDO, Kumamoto, Japan) at a 1 : 25,000 dilution, followed by observation of bright field (BF), GFP, Alexa Fluor 647, and DAPI images using confocal microscopy (A1; Nikon, Tokyo, Japan) with 10× or 20× magnification lenses.

For immunofluorescence staining, tissue sections were washed with 0.1 M PBS for 10 min at room temperature. After rinsing with PBS, tissue sections were blocked with 10% normal goat serum in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature and then incubated with the following primary antibodies overnight at 4°C, rabbit anti-SphK1 (Abcam, Cambridge, MA, United States; used at 1:500), mouse anti-SphK1 (Santa Cruz, United States; used at 1:500), mouse anti-S1PR2 (Santa Cruz Biotechnology, CA, United States; used at 1:500), rabbit anti-GFAP (Abcam, Cambridge, MA, United States; used at 1:1000), and rabbit anti-NeuN (Abcam, Cambridge, MA, United States; used at 1:1000). On the following day, tissue sections were washed three times for 10 min in 0.1 M PBS and then incubated for 2 h at room temperature with donkey Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Abcam, United States; used at 1:500), and goat anti-mouse IgG H&L (Alexa Fluor^® 647, Abcam, United States; used at 1:500) conjugated secondary antibodies. Following this incubation period, tissue sections were washed three times with 0.1 M PBS for 10 min and then mounted with Vectashield DAPI Hardset mounting medium (Solarbio, China). Immunofluorescence staining pictures were captured employing a LSM800 confocal microscope (Zeiss, Germany).

To immunostain MMP2 and CD34, frozen sections of thrombus were stained with anti-MMP2 (ab92536; 1 : 250; Abcam, Cambridge, UK) and anti-CD34 (ab81289; 1 : 200; Abcam). After 1 h incubation at room temperature with fluorescent-labeled secondary antibodies (goat anti-mouse Alexa Fluor® 647; ab150115; 1 : 1000; Abcam), a confocal microscope was used to capture the images under the same conditions for each experiment.

Protein expression and distribution in hPDLSCs and periodontal tissues were investigated using immunofluorescence. Cell samples were rinsed in PBS and fixed with 4% PFA after 2 h of DAP treatment. Next, permeabilized the membrane of cells with 0.5% Triton X‐100 and blocked the samples in PBS containing 1% BSA for 1 h. Following incubation overnight at 4°C with the primary antibody Phospho‐p38 (ZEN BIO), cells were incubated with the fluorescein‐conjugated anti‐rabbit IgG AlexaFluor®647 (Abcam) for 2 h. The cytoskeleton was stained with phalloidin (Thermo Fisher Scientific) and the nuclei was stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI; Sigma). The preparation of tissue slices was identical to that used for immunohistochemistry. Rabbit anti‐CARD4 antibodies (Bioss Antibodies) were used as primary antibodies. Anti‐rabbit IgG (Alexa Fluor® 647, Abcam) was applied as secondary antibodies. Nuclear staining was performed with DAPI (Sigma). The fluorescence staining of cells and tissues was examined using CLSM (FV3000, Olympus, Japan).