A21448

Primary antibodies used for immunoblotting were rabbit anti-GFP (Merck, G1544), rat anti-RFP (Chromotek, 5F8), or mouse anti-GAPDH (ThermoFisher, AM4300). Secondary antibodies for immunoblotting were LI-COR IRDye 680T conjugated goat anti-rat (926–68029), donkey anti-rabbit (926–68023) or goat anti-mouse (926–68020), or LI-COR IRDye 800CW conjugated donkey anti-rabbit (926–32213) or goat anti-mouse (926-32210). Primary antibodies used for immunocytochemistry were anti-TGN46 (Bio-Rad, AHP500G), mouse anti-Paxillin (BD Biosciences, 610051), rabbit anti-Zyxin (abcam, ab71842), and secondary antibodies were Alexa Fluor 647 conjugated donkey anti-sheep (A-21448, ThermoFisher), goat anti-mouse (A-21236, ThermoFisher) or goat anti-rabbit (A-21245, ThermoFisher).

Flow cytometric analysis was done using a FACSVerse, FACSAriaII, or Accuri c6 (BD Biosciences) and sorting was done using FACSAriaII devices (BD Biosciences) at the UCSF Parnassus Flow Cytometry Core; acquisition software used was FACSDiva 6 or higher, or Accuri c6 software v1.0. For the collagen uptake assay, cells were incubated in fresh media with Oregon Green 488-conjugated gelatin (10 μg/ml final concentration; Life Technologies) for 1 h at 37 °C. Cells were then harvested (for suspension cells) or trypsinized and harvested for analysis. To assess only internalized fluorescent collagen (i.e. to ignore bound but not internalized collagen), cells were either washed before flow cytometry or Trypan blue was added to quench bound but not internalized fluorochromes as we have done previously^{21 (link),64 (link)}. For cell-surface staining, cells were stained using a standard FACS staining protocol. Antibodies included: anti-MRC2 (as above) as a primary antibody with a secondary of Donkey anti-sheep conjugated to a fluorophore (1:250, Invitrogen, A-21448). Cells were then analyzed and/or sorted on one of the above devices, with sorting and analysis gates based on fluorescence-minus-one controls after compensation was performed with single-stain controls. Data were analyzed with FlowJo, version 7.6.1 (Tree Star, Ashland, OR).

The frozen sections (n = 3 per group) were first permeabilized using 0.1% Triton X-100 for 10 min followed by blocking with 5% donkey serum in PBS for 30 min. The sections were then incubated with primary antibodies overnight at 4 °C, subsequently rinsed with PBS, and incubated with secondary antibodies at RT for 1 h. After rinsing with PBS for 3 times, the slides were mounted using Vectashield mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). The slides were subsequently imaged under immunofluorescence microscope (Axio Scope.A1, Zeiss, Oberkochen, Germany).
The primary antibodies used in this study included mouse anti-NeuN (1:400, ab104224, Abcam, Cambridge, England), mouse anti-GFAP (1:100, sc-33673, Santa Cruz Biotechnology), rat anti-CD68 (1:200, MCA1957, Bio-Rad Laboratories, Hercules, USA), sheep anti-PGRN (1:200, AF2557, R&D Systems, Minneapolis, USA), and rabbit anti-iNOS (1:200, PA1-036, Invitrogen, Carlsbad, USA). The secondary antibodies used in this study included donkey anti-mouse Alexa Fluor 488 (1:200, A-11017, Invitrogen, Carlsbad, USA), donkey anti-rat Alexa Fluor 488 (1:200, A-21208, Invitrogen, Carlsbad, USA), donkey anti-rabbit Cy3 (1:200, AP182C, Sigma-Aldrich, Darmstadt, Germany), and donkey anti-sheep Alexa Fluor 647 (1:200, A-21448, Invitrogen, Carlsbad, USA)

HepG2 cells were plated at 150,000 cells per well on acid-washed and sterilized glass coverslips in a 12-well plate. Cells were stimulated as described before and then washed at 1, 6, 12, and 24 h in cold PBS (Gibco) and fixed for 10 min in 4% paraformaldehyde (AAJ19943K2, Thermo Fisher Scientific). Cells were blocked in 10% BSA and permeabilized with Triton X-100. 1:600 anti-ATP7B (ab124973, Abcam) and 1:600 anti-TGN46 (GTX74290, GeneTex) antibodies were used to stain the copper transporter and trans-Golgi network marker, respectively, and anti-rabbit IgG AlexaFluor 488 (R37116, Invitrogen) and anti-sheep IgG AlexaFluor 647 (A21448, Invitrogen) were used as secondary antibodies to fluorescently label the proteins. A DAPI dye (R37606, Invitrogen) was used to stain the nucleus, and coverslips were sealed to glass slides along with Prolong Gold Antifade Mountant (P36930, Invitrogen). Fixed and mounted cells were imaged using a laser scanning confocal microscope (Olympus FluoView FV1000) using a ×60 oil immersion lens at the UC Davis Molecular and Cellular Biology Light Microscopy Imaging Facility.

Embryos were fixed overnight with 4% PFA at 4 °C; permeabilized for 10 h in 2% Triton X‐100 in PBS at room temperature; blocked overnight in 0.1% Tween‐20, 0.01% Triton X‐100, and 1% BSA in PBS at 4 °C; and incubated with primary antibodies at 4 °C overnight. The primary antibodies were PAX6 (1:100, AF8150, R&D Systems), CD31 (1:100, ab222783, Abcam), GATA4 (1:100, sc‐25310, Santa Cruz), myosin light chain (1:100, ab79935, Abcam), myosin heavy chain (1:100, 53‐6503‐82, eBioscience). The secondary antibodies were incubated overnight at 4 °C. The secondary antibodies were Alexa 488 goat anti‐rabbit (1:500, A11034, Invitrogen) and Alexa Fluor 647 donkey anti‐sheep (1:500, A21448, Invitrogen). Nuclear staining and incubation in were performed 10 µg mL⁻¹ Hoechst 33 342 (H3570, Invitrogen) for 10 min at room temperature. The embryos were imaged on confocal microscopes Leica SP8, and Zeiss LSM780 and Imaris 9.0.2 software was used to construct the 3D images/videos.