Donkey anti mouse igg

The following antibodies were purchased: anti-FAM83H (HPA024604; Sigma-Aldrich), anti-keratin 8 (TS1; Thermo Fisher Scientific), anti-keratin 18 (DC10; Thermo Fisher Scientific), anti-keratin 19 (RCK108; Thermo Fisher Scientific), anti-CK1α (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CK1ε (HPA026288; Sigma-Aldrich), anti-CK1δ (R-19; Santa Cruz Biotechnology), anti-SON (HPA023535; Sigma-Aldrich), anti-actin (C-11; Santa Cruz Biotechnology), anti-SC-35 (S4045; Sigma-Aldrich), anti-FLAG (M2; Sigma-Aldrich), anti-hnRNP M (GTX114999; GeneTex, San Antonio, TX, USA), and anti-GAPDH (GT239; GeneTex) antibodies. We used MAb104 hybridoma culture fluid to detect phospho-SR proteins. Alexa Fluor 488, 594, and 647 donkey anti-mouse IgG, Alexa Fluor 488, 555, and 594 donkey anti-rabbit IgG, and Alexa Fluor 488, 555, and 647 donkey anti-goat IgG antibodies were used for immunofluorescence (Life Technologies). HRP-conjugated horse anti-mouse IgG (Cell Signaling Technology, Beverly, MA, USA), donkey anti-rabbit IgG (GE Healthcare, Little Chalfont, UK), bovine anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA), and donkey anti-mouse IgM (Jackson ImmunoResearch) antibodies were used for Western blotting.

For IHC, frozen sections were washed with 1X PBS three times followed by incubating with 0.3% H₂O₂/0.3% horse serum in 1X PBS for 5 minutes at RT. Then, slides were washed in TNT buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.05% Tween-20) 3 times. Slides were then blocked one hour with TNB buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.5% blocking reagent) at RT, followed by incubation with primary antibody (COL1A1, Rockland, 1:1000) diluted in TNB buffer overnight at 4°C. Slides were then washed with TNT buffer 3 times, incubated with Horseradish peroxidase (HRP) conjugated secondary antibodies (Life Technologies) for 2 hours at RT, signals were developed by using the DAB staining kit (Vector Laboratories, Cat.# SK-4100). For IF staining, frozen sections were washed in PBS, and then blocked by 5% donkey serum in PBST for one hour at RT, followed by incubation with primary antibodies (α-SMA, 1:3000; COL1A1, Rockland, 1:1000; KLF4, Cell Signaling Technology, 1:50; FBXO32, 1:100, ECM Bioscience; PDGFRB, 1:50, Cell Signaling Technology) overnight at 4°C. Alexa Fluor 488 or 568-conjugated secondary antibodies (Donkey anti-Mouse IgG, or Donkey anti-Rabbit IgG or Donkey anti-Goat IgG; Life Technologies, 1:250 dilution) were used for the visualization of signals. Nikon DS Ri1 and SPOT software were used for the image acquisition and analysis.

Primary myoblasts were put into Matrigel coated-96-well plate at a high density with four replicate wells for each genotype by IGF1 treatment combination. The myoblasts were fused into myotubes for 3 days in fusion media with different concentration (0, 12.5, 25, 50, 100 and 200 ng/mL) of IGF1. The mature myotubes were fixed in 4% formaldehyde in PBS for 5 minutes at room temperature, washed 3 times with PBS and permeabilized with 0.5% TritonX100 for 5 minutes. After washing 3 times with PBS, the cells were blocked for 1 hour in 1% BSA in PBS. Cells were incubated with anti-myosin heavy chain antibody (MF20, Developmental Studies Hybridoma Bank) diluted in 0.1% BSA overnight at 4°C, and washed 5 times in PBS. Secondary antibody (Donkey anti-mouse IgG, Life Technologies) were diluted in 0.1% BSA and filtered through 0.2 μM filters. Cells were incubated in secondary antibody for 1 hour and washed 5 times with PBS. The fluorescence signals were read on Tecan Genios Pro (Tecan Group Ltd., Morrisville, NC USA) plate reader.

Cells were fixed either with 4% paraformaldehyde (PFA) for 20 min or with cold methanol for 5 min at -20°C for proper axoneme proteins detection [22 (link)]. They were then washed and incubated for 30 min in blocking buffer (10% FCS in PBS) followed by incubation with primary antibodies diluted in blocking buffer supplemented with 0.05% saponin for 1 h at room temperature or overnight at 4°C. Cells were washed 3 times, and then incubated for 1 h with fluorescent Alexa-Fluor secondary antibodies. After washing, 150 μl of DAPI-Fluoromount were added into the Luer chamber (Southern Biotech). Images were acquired with a Zeiss Apotome.2 fluorescence microscope equipped with a 63x oil immersion fluorescence objective. Number of ciliated cells and length of cilia were quantified using Zen Software (Zeiss) or Imaris Software (Bitplane). The following antibodies were used for immunofluo-rescence: mouse-anti-LC3B (MBL, Cat# M152-3); rabbit-anti-FLCN (Cell signaling, Cat#3697); mouse-anti-ARL13B (C-5) (Santa Cruz, Cat#515784); rabbit-anti-ATG16 (MBL, Cat#PM040); rabbit-anti IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); Phalloidin (Cat# A34055); Alexa Fluor-conjugated secondary anti-bodies (donkey anti-mouse IgG and donkey anti-Rabbit IgG, Life Technologies).

Immunofluorescence double staining was performed to observe the expression of β2-AR and β-arrestin2 in activated HSCs of HCC. The liver tissue and cell slides were incubated with primary antibodies for 12 h in a dark, humid chamber at 4°C. Antibodies against β2-AR (sc-81577), β-arrestin2 (sc-13140) and alpha-smooth muscle actin (α-SMA) (sc-130617) were obtained from Santa Cruz Biotechnology (CA, USA). The next day, slides were incubated with secondary antibodies: goat-anti-rabbit IgG and donkey-anti-mouse IgG (Life Technologies, New York, USA) at room temperature for 1 h in the dark. Following washing 3 times with 1× PBS, DAPI (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) was used to stain the nuclei at room temperature for 10 min, and stored at 4 °C. Finally, Leica TCS SP8 confocal microscope (Leica Microsystems, Leica, Germany) was used to observe and capture fluorescent sections.

Vascular tissues were harvested, washed using PBS and fixed for 6–8 h at 4 °C in 4% paraformaldehyde. Then, tissues were dehydrated in 30% sucrose solution overnight at 4 °C, embedded in OCT and frozen at − 80 °C. Frozen tissues were sliced into 10-μm thick sections using a cryostat (Leica, CM1950). Immunofluorescence staining was performed as described [27 (link)]. The primary antibodies used in this assay were αSMA antibody (Sigma, F3777, 1:500), SM22 (Abcam, ab14106, 1:200), CNN1 (Abcam, ab46794, 1:200), SMMHC (Abcam, ab53219, 1:200), RFP antibody (Rockland, 600-401-379, 1:50) and Ki67 (Abcam, ab53219, 1:200). The Alexa Fluor-conjugated secondary antibodies used in this study were Donkey anti-Mouse IgG (Invitrogen, A21202 for Alexa Fluor 488), Donkey anti-Rabbit IgG (Invitrogen, A31572 for Alexa Fluor 555), Donkey anti-Rabbit IgG (Invitrogen, A32731 for Alexa Flour 488) and Donkey anti-goat IgG (Invitrogen, A32816 for Alexa Fluor 555). Images were taken using the Nikon A1 confocal microscope. The co-staining area was quantified using the colocalization tool of Fiji. The mean gray value was calculated using the Fiji measurement tool.