Alexafluor 488 conjugated goat anti mouse igg1

We performed double-immunofluorescence staining for LC3B with ubiquitin, p62 with ubiquitin and LC3B with p62 to investigate a possible co-localization of them in the same inclusion. FFPE tissue sections were cut at 1 μm, dewaxed, rehydrated and pretreated with Target Retrieval Solution (Dako) at pH 9.0 for 20 min at 97 °C. We used the primary antibodies anti-LC3B (#3868, Cell Signaling), anti-p62 (#BWL PW9860, Enzo, Life Sciences, NY, USA), anti-p62 (#Sc-28359, Santa Cruz) and anti-ubiquitin (#NB300-130, Novus, Littleton, CO). The secondary antibodies used were Cy3-conjugated goat anti-rabbit IgG (H+L) (#111-166-045, Dianova, Hamburg, Germany) and Alexa Fluor 488-conjugated goat anti-mouse IgG1 (#A21121, Thermo Fisher Scientific). Double-labeling for LC3B with ubiquitin: anti-LC3B antibody (1:20) was labeled with Cy3 (1:100) and anti-ubiquitin antibody (1:1000) with Alexa Fluor 488 (1:100), each incubation was performed twice for 30 min at RT. Double-labeling for p62 with ubiquitin: anti-p62 (Enzo) antibody (1:500) was labeled with Cy3 (1:800) and anti-ubiquitin antibody (1:1000) with Alexa Fluor 488 (1:100). Double-labeling for LC3B with p62: anti-LC3B antibody was labeled with Cy3 and anti-p62 (Santa Cruz) antibody with Alexa Fluor 488 (for details, see S4 Table). DNA was stained with DAPI and images were analyzed as described above.

Paraffin sections of liver tissue (5 μm) were stained with haematoxylin and eosin (H&E) for light microscopy. Five random sections were investigated per slide to measure the necrotic area and determine the percentage of apoptotic cells. To measure the necrotic areas, sections were observed under an Axiovert 40 CFL microscope (Carl Zeiss, Oberkochen, Germany) and measured using iSolution DT 36 software (Carl Zeiss). For immunofluorescence staining, after deparaffinisation, sections were immunostained with antibodies against F4/80 (ab6640), Ly6G (ab25377), and 4-hydroxynonenal (4-HNE, ab46545) (all from Abcam). TUNEL staining was performed with commercial kits (Promega). After washing with phosphate buffered saline (PBS), secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG1 and Alexa Fluor 594-conjugated goat anti-rabbit IgM, Thermo Fisher Scientific) were incubated for 1 h at 37 °C. Sections were counterstained with DAPI.

Liver tissues were fixed in 10% formaldehyde solution at 4 °C for paraffin sections or placed in 30% sucrose solution and embedded with liquid nitrogen-cooled isopentane for frozen sections. Paraffin sections of liver tissue were stained with hematoxylin and eosin (H&E) for light microscopy. For Oil Red O staining, frozen sections were incubated with the dye for 15 min and washed with 1× PBS. For immunofluorescence analysis, primary hepatocytes were stained overnight at 4 °C with antibodies against NCoR1 (Cell Signaling Technology) or PPARα (Santa Cruz Biotechnology). After washing with PBS, the sections were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG1 and Alexa Fluor 594-conjugated goat anti-rabbit IgM; Thermo Fisher Scientific) for 1 h at 37 °C and then counterstained with DAPI. NCoR1 and PPARα interactions were detected using a Duolink Proximity Ligation Assay kit (#DUO96010, Sigma-Aldrich).

Immunostaining and alkaline phosphatase (AP) staining were performed as described previously [15 (link)]. Primary antibodies were anti-stage-specific embryonic antigen 1 (SSEA1, MC-480; Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA), anti-Oct3/4 (MBL, Nagoya, Japan), anti-nestin (Rat-401; DSHB), anti-α-smooth muscle actin (Progen, Heidelberg, Germany), anti-neuronal class III β-tubulin (Tuj1; Covance, Richmond, CA), anti-Sox2 (Millipore, Bedford, MA), anti-glial fibrillary acidic protein (GFAP; Dako, Carpenteria, CA), anti-α-actinin (Sigma-Aldrich, Tokyo, Japan), anti-α-fetoprotein (R&D Systems, Minneapolis, MN), anti-Sox1 (Cell Signaling Tech, Beverly, MA), anti-Sox10 (Millipore), anti-PGP9.5 (Ultra Clone, Wellow, Isle of Wight, UK), and anti-GFP (Nakarai Tesque, Kyoto, Japan). Secondary antibodies were Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG1, Alexa Fluor 568-conjugated goat anti-mouse IgM, Alexa Fluor 647-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated goat anti-rat IgG, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (all purchased from Molecular Probes, Life Technologies, Eugene, OR).

Primary antibodies used were as follows: mouse monoclonal IgG2b anti-IL-10 (1:1,000 dilution; Cat. No. SC-365858; Santa Cruz Biotechnology; Santa Cruz, CA, USA); rabbit polyclonal IgG anti-Chicken IL-17 (1:2,000 dilution; Cat. No. LS C294849; Lifespan Biosciences; Seattle, WA, USA); mouse monoclonal IgG2b anti-claudin-1 (1:300 dilution; Cat. No. SC-166338; Santa Cruz Biotechnology; Santa Cruz, CA, USA); and rabbit polyclonal IgG anti-Rat TJP1/ZO-1 (1:1,500 dilution; Cat. No. LS C145545; Lifespan Biosciences; Seattle, WA, USA). Primary antibodies were detected using the following secondary antibodies (1:10,000 dilution; Invitrogen, Thermo Fisher Scientific Inc.; Waltham, MA, USA): Alexa-Fluor 488 conjugated goat anti-mouse IgG1 (Cat. No. A-11008) and Alexa-Fluor 555 Plus conjugated goat anti-mouse IgG (Cat. No. A-32727).