Alexa fluor 488 594 conjugated secondary antibodies

The kidney, liver, lung, and heart were embedded in paraffin as per standard protocol, sectioned into 4–6 μm thin sections, and then the tissues were stained with H&E, periodic acid-Schiff (PAS), Masson’s trichrome, Sirius Red, or immunohistochemically stained with primary antibodies against collagen type I (1:500, rat, Abcam) and α-smooth muscle actin (1:1000, rat, Abcam). A light microscope (Olympus, Japan) was used to observe the morphological structure of each tissue for histological analysis as described in our previous study [4 (link)].
For immunofluorescence analysis, the frozen tissues were cut into 6 μm sections and incubated for 14 h at 4 °C with primary antibodies against insulin (1:200, guinea pig, Abcam), glucagon (1:2000, mouse, Abcam), F4/80 (1:100, rat, Santa Cruz), iNOS (1:100, rat, Abcam), Arg (1:100, rabbit, Abcam), collagen type I (1:500, rabbit, Abcam), and α-smooth muscle actin (1: 100, mouse, Sigma-Aldrich), followed by incubation with Alexa Fluor 488/594-conjugated secondary antibodies (1:500, Invitrogen, USA) at room temperature for 2 h. The nuclei were stained with DAPI. The immunofluorescently stained sections and cells were examined and photographed using a laser scanning confocal microscope (Leica, Wetzlar, Germany).

HEY cells were fixed for 15–20 min in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (in PBS) for 10 min. After three PBS washes, cells were blocked with 2% BSA for 30 min at room temperature. Cells were then incubated with anti-USP13 antibody (Santa Cruz, sc-514416, 1:500) and anti-MCL1 antibody (Santa Cruz, sc-819, 1:500) diluted in 2% BSA at 4 °C overnight. After three PBS washes, the cells were incubated with 1 μg mL⁻¹ Alexa Fluor 488/594-conjugated secondary antibodies (Invitrogen) shaking at room temperature in the dark for 1 hour. Cells were washed three times with PBS in the dark, stained with DAPI (Invitrogen) and mounted in Prolong^® Gold Antifade Reagent (Invitrogen). For mitochondrial morphology analysis, SW-1573 and TOV-21G cells were grown on coverslips inside a Petri dish and incubated with pre-warmed staining solution containing 50 nM MitoTracker Red CMXRos (Invitrogen) for 45 min at 37 °C. The immunofluorescent staining was observed using a confocal microscope (Leica).

The mouse pancreatic tissues were harvested 1, 2, or 4 weeks after MSC infusion. First, the mice anaesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) were perfused with PBS through the left ventricle, followed by 4% paraformaldehyde. Then, the pancreases were isolated, dehydrated with 30% sucrose/PB overnight, and embedded in optimal cutting temperature compound (OCT). Pancreatic sections (6 mm) were sliced by a microtome (Thermo Fisher Scientific) and incubated in a humidified chamber at 4°C overnight with primary antibodies against insulin (1/200, guinea pig, Sigma-Aldrich), glucagon (1/2,000, mouse, Abcam), Pdx1 (1/200, rabbit, CST), CD11c (1/200, mouse, Abcam), IL1β (1/100, rabbit, Abcam), F4/80 (1/200, rabbit, Sigma-Aldrich), and Fizz1 (1/200, rabbit, Abcam). After the sections were washed with PBS, they were incubated for 2 h with a secondary antibody (1 : 500; Alexa Fluor 488/594-conjugated secondary antibodies, Invitrogen) at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). The images were captured with a confocal laser scanning microscope (Olympus, Tokyo, Japan). Peritoneal macrophages spread on glass coverslips were fixed with 4% paraformaldehyde. The remaining steps were performed as described above.

The Cell Titer Glo cell viability assay kit was purchased from Promega. RNAiMAX and Lipofectoamine 2000 were obtained from Life Technologies. Opti-MEM was purchased from GIBCO. RPMI and DMEM media were purchased from Cellgro. Pre-designed small interfering RNA (siRNA) directed against human MAGE-A12 (silencer select, siRNA#1 (s8451): 5’-CACUCUAUUCUGUAAAUUU-3’, siRNA#2 (s8453): 5’-GGAGACGAGCUUCCAAGUA-3’), CDKN1A (p21, siRNA#1: s417), and a negative scramble control (#1) were purchased from Life Technologies. MG132 was purchased from Calbiochem. Antibodies against MAGE-A12 (mouse monoclonal, ab87973, Abcam), p53 (Ab-6, Merck), Cdk1 (610037, BD), Cdk2 (610145, BD), phospho-cdc2 (Y15) (#9111, CST), cdc25C (#4688, CST), alpha-tubulin (T5168, Sigma), FLAG (M2, Sigma), HA (3F10, Roche), Myc (9E10, Roche), beta-actin (Sigma), horseradish-peroxidase (HRP)-conjugated secondary antibodies (GE Health Care), and Alexa Fluor 488/594-conjugated secondary antibodies (Life Technologies) were purchased from the indicated sources.

Mice were anaesthetized with intraperitoneal injection of sodium pentobarbital and then underwent consecutive perfusions of 20 ml of PBS and 20 ml of 4% paraformaldehyde through the right ventricle. For H&E staining, EAT was embedded in paraffin, sectioned at 10 μm thickness and then stained with H&E according to the standard protocol. For immunofluorescence staining, EAT was dehydrated through incubation in 30% sucrose/PB overnight, embedded in optimal cutting temperature compound (OCT, Sakura, Finetek, USA) and cut into frozen sections at 10 μm. The frozen sections were blocked with BSA and then incubated at 4 °C overnight with indicated primary antibodies against Fizz1 (ab39626, Abcam, San Francisco, USA), iNOS (ab15323, Abcam, San Francisco, USA) and F4/80 (sc-377009, Santa Cruz, USA), followed by further incubation with Alexa Fluor 488/594-conjugated secondary antibodies (1: 500, Life Technologies), shielding from light, at room temperature for 2 h. Finally, the sections were incubated with 4′-6-diamidino-2-phenylindole (DAPI) for 7 min for nuclear staining and imaged with a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Anti-Rab11 mouse monoclonal antibody (#610657, BD Transduction Laboratories, Lexington, KY), anti-TfR mouse monoclonal antibody (#13–6800, ZYMED Laboratories Inc., San Francisco, CA), anti-β-actin mouse monoclonal antibody (#G043, Applied Biological Materials Inc., Richmond, BC, Canada), anti-green fluorescent protein (GFP) rabbit polyclonal antibody (#598, MBL, Nagoya, Japan), anti-TBC1D12 rabbit antibody (#HPA038277, Sigma-Aldrich, St. Louis, MO), horseradish peroxidase (HRP)-conjugated anti-FLAG tag (M2) mouse monoclonal antibody (#A8592, Sigma-Aldrich), HRP-conjugated anti-T7 tag mouse monoclonal antibody (#69048-3CN, Merck KGaA, Darmstadt, Germany), HRP-conjugated anti-GFP rabbit polyclonal antibody (#598–7, MBL), and Alexa Fluor 488/594-conjugated secondary antibodies (Thermo Fisher Scientific Corp., Hudson, NH) were obtained commercially. Anti-LC3β (simply referred to as LC3 throughout this paper) rabbit polyclonal antibody was affinity-purified as described previously [19 (link)]. Rab11A/B siRNAs were prepared as described previously [11 (link)]. Stealth RNAi^™ siRNAs (simply referred to as siRNAs hereafter) against rat TBC1D12 (target site: 5’-AAUACUUCAACAUCAUGCUGUGAUC-3’ [siTBC1D12 #1] and 5’-AUUAAUUCCAGACUAGCCUCUCGGU [siTBC1D12 #2]) were purchased from Life Technologies.