Alexa fluor 488 conjugated anti rabbit igg

The slides were incubated overnight at 4°C with the primary antibody, washed in PBS and incubated with secondary antibodies for 1.5 hours at room temperature. This was followed by washing with PBS and mounting with Vectashield‐DAPI mounting medium. Primary antibodies used in our experiment were as follows: mouse anti‐RET antibody (1:100; Santa Cruz, Santa Cruz, CA) and rabbit anti‐ChgA antibody (1:200; Abcam). Secondary antibodies were Alexa Fluor 647‐conjugated anti‐mouse IgG (1:400; Abcam) and Alexa Fluor 488‐conjugated anti‐rabbit IgG (1:400; Abcam), and DAPI (Abcam). Images were taken on Zeiss LSM 880 with an AiryScan confocal laser scanning microscope and were analysed with zen 2009 Light Edition software.

Cells were plated in the chamber-slides in the same conditions, and treated with compound 8a, gemcitabine or their combination for a further 48 h. Cells were washed twice with PBS and fixed with paraformaldehyde (4%) at room temperature for 15 min. After washing twice with PBS, cells were blocked in PBS containing 0.2% Triton X-100, 10% goat serum and 5% BSA for 2 h, and then incubated with primary antibodies (γ-H2AX, Ser139, Cell Signaling Technology, Cat#9718) overnight at 4 °C. Cells were stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077) for 2 h at 4 °C in dark. After washing with PBS, the cells were stained with Hoechst and observed by confocal microscopy (Leica TCS SP8 STED 3X).

The rabbit polyclonal anti-SPARC antibody (15274-1-AP) was purchased from Proteintech. The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology. The mouse monoclonal anti-human cath-D antibody (clone 49, #610801) was purchased from BD Transduction Laboratories^TM, and the goat polyclonal anti-mouse cath-D (AF1029) from R&D Systems. The anti-human cath-D antibodies M1G8 and D7E3 were previously described 19 (link). The mouse monoclonal anti-tubulin antibody (clone 236-10501, #A11126) was from Thermo Fisher Scientific, the mouse monoclonal anti-Myc (clone 9B11) from Ozyme, and the rabbit polyclonal anti-β actin antibody (#A2066) from Sigma-Aldrich. The horse anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (#7076), and goat anti-rabbit IgG-HRP (#7074S) secondary antibodies were from Cell Signaling Technology. The donkey anti-goat HRP conjugated antibody (FT-1I7890) was from Interchim. The Alexa Fluor 488-conjugated anti-rabbit IgG (#Ab150077) was purchased from Abcam, and the Cy3-conjugated anti-mouse IgG (#SA00009.1) from Proteintech. Hoechst 33342 (#FP-BB1340) was from Interchim FluoProbes.

The PCAGGS-HSP90A-His and pcDNA3.1-KNG1-3 × Flag or pcDNA3.1-MAVS-Flag plasmids were transfected into HepG2.2.15 cells, which were grown on coverslips for 48 h. HepG2.2.15 cells were washed three times with PBS before fixation with 4% formaldehyde for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and then blocked with 5% bovine serum albumin for 1 h. Cells were incubated with the corresponding antibodies for 1 h at room temperature. After washing three times with TBST, cells were incubated for 1 h at room temperature with Alexa Fluor® 488 conjugated anti-rabbit IgG (#ab150077, Abcam) and Alexa Fluor® 647 anti-mouse IgG (#ab150115, Abcam). DAPI (#P0131, Beyotime) was used for nucleus staining. The intracellular localization was analyzed by a confocal microscope (Nikon, Tokyo, Japan).

Pancreatic tissue sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: anti-Glucagon (Cell Signaling Technology, Cat#2760), anti-insulin (Cell Signaling Technology, Cat#3014). The sections were stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077) for 2 h at 4 °C in dark. After washing with PBS, the sections were stained with DAPI and observed by confocal microscopy (Leica TCS SP8 STED 3X).

hDPCs were rinsed with PBS and fixed with 4% paraformaldehyde in PBS at 4 °C for 10 min. Fixed cells and sections were permeabilized with PBS containing 0.1% Triton X-100 and then blocked with 10% normal donkey serum for 30 min. The samples were incubated overnight with primary antibodies: anti-TRAF6 (1:500, GTX113029, polyclonal, rabbit; GeneTex), anti-IRAK1 (1:500, GTX31253, polyclonal, rabbit; GeneTex), and anti-RELA (1:1000, D14E12, monoclonal, rabbit; Cell Signaling Technology). Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, donkey; Abcam, Cambridge, UK) was applied for visualization. Coverslips were mounted with a fluorescent mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Flouroshield Mounting Medium with DAPI; Abcam). A confocal laser scanning microscope (Leica TCS-SP8; Leica Microsystems) and LAS AF confocal software (Version 1.8.3; Leica Microsystems) were used to perform histological assessment.