Alexa fluor 594 goat anti mouse igg

Isolated hepatocytes or LSECs were cultured on a coverslip (AGC Techno Glass Co., Shizuoka, Japan), fixed with paraformaldehyde for 5 min and blocked with SuperBlock blocking buffer (Thermo Fisher Scientific). The cells were incubated with an E-cadherin antibody (BD Biosciences, San Jose, CA, USA) and VE-cadherin antibody (Abcam) at 1:200 for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were used as secondary antibodies for 1 h at room temperature with protection from the light. The cells were counterstained with DAPI and analyzed by fluorescence microscopy (Keyence Corporation, Osaka, Japan).
Mouse livers were mounted in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co Ltd., Tokyo, Japan) and frozen until preparation. Frozen sections were cut at 10-μm thickness and fixed in methanol for 10 min. Blocking was performed in PBS with 3 % bovine serum albumin (BSA). The slides were incubated with BMPER antibody (Abcam) at 1:200 in PBS overnight at 4 °C, and subsequently incubated with CD31 antibody (BD Pharmingen) at 1:100 in PBS for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were incubated at 1:200 in PBS for 1 h at room temperature. The slides were counterstained with DAPI and analyzed using fluorescence microscopy.

Tissues were fixed upon slides with acetone, and then permeabilized with 0.1 µg/ml Triton X-100 in PBS. Tissues were incubated in 2.5% normal horse serum (Vector, Burlingame, CA, USA), followed by treatment with rabbit anti-ET-B antibody (Abcam Inc., Cambridge, MA, USA; 1:4000 dilution) and mouse anti-PMEL17 antibody (DAKO; 1:40). Tissues were incubated with Alexa Fluor^® 488 goat anti-rabbit IgG and Alexa Fluor^® 594 goat anti-mouse IgG (both from Life Technologies; 1:500), followed by nuclear staining with 4′6-diamidino-2-phenylindole (DAPI) (Life Technologies; 1:2000). Slides were mounted in Fluoromount-G^® (Southern Biotech, Birmingham, AL, USA). Co-cultured NHEKs and NHEMs in 4-well culture slides were fixed with 4% paraformaldehyde in PBS and were quenched by 50 mM NH₄Cl in PBS. The cells were then permeabilized with 50 µg/ml digitonin in PBS and were then incubated in PBS containing 0.2% gelatin, followed by treatment with rabbit anti-p62 antibody (MBL; 1:500) and mouse anti-PMEL17 antibody (DAKO Inc.; 1:500). Cells were then incubated with Alexa Fluor^® 488 goat anti-rabbit IgG and Alexa Fluor^® 594 goat anti-mouse IgG (both from Life Technologies; 1:1000). Slides were mounted in ProLong^® Gold Antifade Reagent with DAPI (Life Technologies), and images were obtained with a Leica DM5500B digital microscope (Leica Microsystems, Bannockburn, IL, USA).

For antigen co-localization studies, double-fluorescence immunostaining was performed using a sequential method. After deparaffinization and antigen retrieval, blocking for non-specific binding was performed at 4°C overnight. Appropriate concentrations of antibodies were then sequentially applied for 1 hour at room temperature, with PBS washing after each incubation. Slides were first incubated with anti-CD45, anti-CD68, CD20 or CD3 (IR751, IR7613, IR604 and IR503 without further dilution, Dako), followed by incubation with Alexa Fluor 594 goat-antimouse IgG (diluted 1/200; Life Technologies). Next, slides were incubated with the anti-15-PGDH antibody followed by incubation with Alexa Fluor 488 goat-antirabbit IgG (diluted 1/200; Life Technologies). As a negative control, sections were incubated omitting primary antibodies. Samples were then mounted with ProLong Gold antifade reagent with DAPI (Molecular Probes, Life Technologies Co, Eugene, OR). Images were obtained using an SP5 Leica confocal microscope.