Alexa fluor 647 anti mouse igg

A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody (#ab2785, Abcam, USA) in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., USA) in 2% BSA at room temperature for 1 hour.
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).

Hybridoma supernatant samples were screened according to an automated FCM method (Wang et al., 2018 (link)). 300-19 cells over-expressing human or mouse SORT1 were labeled with CellTrace Violet (Thermo Fisher Scientific) and Vybrant CFDA SE Cell Tracer (Thermo Fischer Scientific), respectively, following manufacturer’s protocol. The cells were resuspended in cold PBS containing 2% fetal bovine serum (FCM buffer) and incubated with supernatant samples for 30 min at 4°C. After three rounds of washing using cold FCM buffer, 30 μL of Alexa Fluor 647 Anti-Mouse IgG (Jackson ImmunoResearch) was added. After 30 min of incubation at 4°C, the cells were washed twice with FCM buffer, and the binding of antibody was read on an iQue Screener PLUS (IntelliCyt).

Galectin-3 staining was performed according to the protocol described previously^{13 (link)}. Briefly, cells were fixed by treatment with 4% paraformaldehyde/PBS at 22 °C for 15 min and washed twice in PBS for 5 min each. Fixed cells were permeabilized by treatment with 0.1% Triton-X 100 in PBS at 22 °C for 10 min and then incubated with 2% BSA/PBS at 22 °C for 30 min. After blocking, cells were incubated with 10 μg/mL Mouse anti-Galectin-3 Monoclonal Antibody (Abcam) in 2% BSA/PBS at 4 °C overnight. Cells were washed three times in PBS for 10 min each and then incubated with 10 μg/mL Alexa Fluor 647 anti-Mouse IgG (Jackson ImmunoResearch Laboratories) in 2% BSA/PBS at 22 °C for 1 h. Thereafter, cells were washed three times in PBS for 10 min each and then mounted with DAPI mounting medium (Abcam). Stained cells were analyzed by using a Nikon fluorescent microscope and quantified by using ImageJ software.