Fluorescent secondary antibody

Cells were cultured on a glass-bottomed cell culture dish (801002, NEST, Wuxi, Jiangsu, China) in the indicated medium and infected with Y. pestis as described above (MOI = 40 for 4 h) or treated with CCCP at the indicated time points. Cells were then washed with PBS and fixed for 15 min at room temperature with 4% paraformaldehyde in PBS, followed by three washes with PBS. After permeabilization with 0.2% Triton X-100 in PBS for 10 min and blocking with 5% BSA in PBS for 1 h, cells were incubated with primary antibodies, including anti-LC3B (1:200, 3868, Cell Signaling Technology, Danvers, MA), anti-COX IV (1:500, ab33985) and anti-Ubiquitin (1:500, ab223378) (Abcam, Boston, MA), and anti-COX IV (1:500, 4850, Cell Signaling Technology) overnight at 4°C. Cells were then washed three times with PBS and incubated for 1 h at room temperature with fluorescent secondary antibody (Cell Signaling Technology), followed by three washes with PBS, incubation with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and observation with the Olympus FluoView FV1000 confocal microscope or the Zeiss LSM 880 confocal microscope.

The cells were incubated with the GSDMD-N antibody (Cell Signaling Technology, 36425) and then with the fluorescent secondary antibody (Cell Signaling Technology). Images were captured using a confocal microscope (Olympus, Tokyo, Japan).

To analyze whether combined SiNPs and B[a]P exposure influences the expression of the G2/M DNA damage checkpoint and apoptosis regulators, we measured the protein levels of Chk1, Cdc25C, cyclin B1/Cdc2, Bcl-2, Bax, Caspase 9, and Caspase 3 in HUVECs, using western blot analysis. The cells were lysed through the RIPA buffer on ice. Equal amounts of protein lysates were loaded onto SDS-polyacrylamide gel electrophoresis, to be separated, and were then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA, USA). 5% skim milk mixed with Tris-buffered saline (TBS) was used to block the PVDF membranes for 1 h. Then, the PVDF membranes were cultured with the appropriate primary antibodies (Cell Signaling Technology, Beverly, MA, USA) at 4 °C for the whole night. The PVDF membrane was washed with TBST three times and was incubated with fluorescent secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) for 1 h in the dark. After having been rinsed with TBST, blotted proteins were detected and imaged through the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincon, NE, USA). Data were analyzed using the Image J software (National Institutes of Health, Bethesda, MD, USA).

The brains were dissected and fixed in paraffin. Coronal cryostat sections (20 μm) were stained with HE and immunofluorescent dyes. Brain sections were fixed at 4°C for 24 hours in 4% paraformaldehyde in PBS (0.01 M, pH 7.4), dehydrated in a series of graded alcohol dehydrations, and fixed in paraffin. The tissues were sectioned (5 μm) with a Leica® RM1850 rotary microtome (Leica Microsystems, Germany). The paraffin sections were dried at 60°C, dewaxed, and subjected to antigen retrieval. The sections were exposed for 1 hour at 37°C to primary antibodies targeting the following proteins: LCN2 (Abcam, 1 : 200), GFAP (Proteintech, 1 : 200), p-JAK2 (Tyr1007/1008) (Abcam, 1 : 100), and p-STAT3 (Tyr705) (Cell Signaling Technology, 1 : 200). The sections were washed twice with ice-cold PBS and then saturated with fluorescent secondary antibodies (Cell Signaling Technology, 1 : 100) in the dark for 1 hour. DAPI (4′,6-diamidino-2-phenylindole) (1 : 1000) was added in the dark for 2 min. The DAPI was rinsed 3 times with PBS for 1 min. The staining procedure for the cells was the same as above. For HE staining, sections were stained with alum HE. Colour images were obtained using a 20x laser scanning confocal microscope (Olympus FV1200, Tokyo, Japan).