Alexa fluor 488 conjugated donkey anti rabbit igg

Immunofluorescence analyses were performed on liver tissues and LX-2 cells treated with HNK or vehicle control essentially as described in Supplementary Materials and Methods. Briefly, cells were incubated on a glass chamber slide with the indicated drug, covered, and incubated with ice-cold 100% methanol for 10 min at −20 °C. After PBS washes, cells were blocked with diluted donkey serum for 30 min at room temperature. Rabbit anti-αSMA (DAKO Agilent Technologies, Santa Clara, CA, USA), rabbit anti-p62 (Progen, Heidelberg, Germany), rabbit anti-SMAD2/3 (Cell Signaling Technology, Danvers, MA, USA), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA) antibodies were used as the primary and secondary antibodies, respectively. Detailed information about antibodies used is available in the Supplementary Materials and Methods section. After washing with PBS, slides were mounted with medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). A BZ-X800 microscope was used for immunofluorescence analyses.

HGF was from R & D (Minneapolis, MN). 5’-Iodoresiniferatoxin (I-RTX), doxorubicin, crizotinib and SB366791 were from Tocris (R & D, Minneapolis, MN); rabbit monoclonal antibodies to, ERK1/2, CREB, pERK1/2, pCREB, p-Met, CAMKIIαand mouse monoclonal antibodies to c-Met, HRP-conjugated horse anti-mouse IgG and goat anti-rabbit IgG antibody were from Cell Signaling Technology (Danvers, MA); bafilomycin A1, rabbit polyclonal antibodies to TRPV1 and HGF, the goat polyclonal antibody against CGRP, and HRP-conjugated mouse monoclonal antibody against β-actin were from Abcam (Cambridge, UK); Alexa fluor 488-conjugated donkey anti-rabbit IgG and Alexa fluor 680-conjugated donkey anti-goat IgG and rhodamine red-X (RRX)-conjugated donkey anti-goat IgG antibody were from Jackson Laboratory (Bar Harbor, ME); Rhodamine phalloidin was from Life Technologies (Carlsbad, CA); and HRP-conjugated goat anti-chicken antibody was from Santa Cruz Biotechnology (Dallas, TX). Anti-a3V-ATPase antibody was a kind gift from Dr. Sun-Wada, Doshisha Women’s College, Japan.

The liver tissues were embedded in OCT and stored at -80°C. OCT-embedded liver tissues were cut into 6μm sections. The sections were fixed in 4% paraformaldehyde for 15 min and then washed with PBS. Next, the sections were incubated in permeabilization solution (0.3% Triton X-100 in PBS) for 15 min and then washed with PBS. Then, the sections were incubated with anti-collagen I (ab34710, Abcam), anti-Ki67 (ab15580, Abcam) or α-SMA (ab7817, Abcam) antibodies. After washing, the sections were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (711-545-152, Jackson ImmunoResearch Labs), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (ab150074, Abcam) or Alexa Fluor 594-conjugated donkey anti-mouse IgG (715-585-150, Jackson ImmunoResearch Labs). The sections were washed with PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI) (10236276001, Roche) and mounted with a cover glass. The images were captured with a confocal laser microscope setup (LSM980, Zeiss) and processed using ZEN (Zeiss).

At the end of experiments in monkeys F and M, several electrolytic lesions were made by passing a direct current through a tungsten electrode (tip negative, 10–20 μA for ∼60 s, 800–1000 μC). Then, the animals were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused transcardially with 0.1 m PBS followed by 3.5% paraformaldehyde. The brains equilibrated with 30% sucrose in PBS were cut in the coronal plane at 50 μm thickness. Every 10th section was stained with cresyl violet, and the recording locations were reconstructed based on electrode tracks and electrolytic lesions.
To visualize the immunoreactive signals of eNpHR3.0-EYFP and NeuN, the sections were immersed in 1% skim milk for 1 h and incubated overnight with rabbit anti-green fluorescent protein antibody (1:1000 dilution; Thermo Fisher Scientific) and mouse monoclonal anti-NeuN antibody (1:1000 dilution; Millipore) in PBS containing 1% normal donkey serum. The sections were then incubated in the same fresh medium containing Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:400 dilution; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-mouse IgG (1:400 dilution; Jackson ImmunoResearch). Images of the sections were digitally captured using an optical microscope equipped with a high-grade CCD camera (model BX-900, Keyence).