Coralite488 conjugated goat anti mouse igg

The suspended cells were fixed with 4% paraformaldehyde for 30 min and washed by centrifugation in PBS. Then cells were resuspended in PBS containing 0.3% Triton X-100 and 5% BSA, and blocked for 1 h. After centrifugation and washing, the cell pellet was resuspended in 3% BSA solution with primary antibodies at room temperature for 1 h. After that, cells were washed twice with PBS. Then the cell pellet was resuspended in 3% BSA solution with the secondary antibodies at room temperature for 1 h. After centrifugation and washing, the resuspended cells were stained with a 1:1 mixture of DAPI solution and 3% BSA solution for 10 min. After washing with the PBS, the sample was standby for observation. The primary antibodies used in this study were CD45 antibody (Cell Signaling Technology, Danvers, MA, USA), EpCAM antibody (Cell Signaling Technology), and cytokeratin 19 antibody (Abcam, Cambridge Science Park, UK). The secondary antibodies were coraLite488 conjugated goat anti-mouse IgG (Proteintech, Chicago, IL, USA), and coraLite594 conjugated goat anti-rabbit IgG (Proteintech). Fluorescence images were taken using a fluorescence microscopy (Olympus, Tokyo, Japan) with a 40× objective lens.

Bovine mammary gland tissue blocks (approximately 2 mm³) were made into paraffin sections using conventional paraffin slice techniques. The morphological changes between normal bovine mammary gland tissue and mastitis tissue were evaluated by conventional H&E staining. The correlation between SYK and TLR4 was analyzed in bovine mammary gland tissue via dual fluorescence staining: Permeabilized sections were blocked using 5% BSA for 1 h; SYK expression was significantly down-regulated in both mammary gland tissue Fluorescent antibodies: CoraLite488-conjugated Goat Anti-Mouse IgG (Cat#SA00013-1, Proteintech Group, Wuhan, China), Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (Cat#SA00009-2, Proteintech Group, Wuhan, China).

Cells were inoculated in a confocal dish and fixed after drug treatment. For DLAT immunofluorescence experiments, the cells were incubated with 100 nM Mitotracker Red CMXRos (M7512, Thermo Fisher Scientific) for 30 min prior to fixation. Cells were permeabilized with 0.3% TritonX-100 for 5 min, blocked with 3% BSA for 30 min at RT, and incubated with primary antibodies overnight at 4 °C. On the second day, cells were incubated with fluorescent-labeled secondary antibodies for 1 hour. Nuclei were stained with DAPI for 15 min. Finally, fluorescent images of the cells were obtained using a Nikon A1Silaser scanning confocal microscope (Nikon Instruments, Inc., Japan). The following antibodies are used for specific molecular markers: Anti-PPP1R15A (10449-1-AP, Proteintech), Anti-DLAT (4A4-B6-C10, Cell Signaling Technology), Anti-EIF2S1/EIF2A (11170-1-AP, Proteintech), CoraLite594–conjugated Goat Anti-Mouse IgG(H + L) (SA00013-3, Proteintech), CoraLite594–conjugated Goat Anti-Rabbit IgG (H + L) (SA00013-4, Proteintech), CoraLite488-conjugated Goat Anti-Mouse IgG(H + L) (SA00013-1, Proteintech), and CoraLite488-conjugated Goat Anti-Rabbit IgG(H + L) (SA00013-2, Proteintech) were used.