Donkey anti rabbit alexa488

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. Primary antibodies for phospho-SMAD3 (1/500; ab52903; Abcam) and YAP-TAZ (1/1000; 93,622; Cell Signaling Technologies) were incubated one hour. Phalloidin staining was achieved simultaneously (Alexa 594 Phalloidin, A12381, ThermoFisher Scientific) Secondary antibodies used were Alexa 488- donkey anti-rabbit (Jackson Immunoresearch Laboratories, West Grove, PA; 1:500). Nuclear staining was achieved by 20 min incubation at room temperature in Hoechst 33,342 (ThermoFisher Scientific). No cellular autofluorescence and no nonspecific labeling were detected in these conditions. Images were collected by confocal microscopy (Zeiss LSM8) and processed using ZEN (Zeiss) and ImageJ software.

For immunofluorescence double-staining, sections were deparaffinized with xylene and ethyl alcohol and quickly rinsed with TBST buffer for 5 min. After blocking with 5% normal serum-TBST for 1 h, sections were incubated with mouse monoclonal anti-CD4 (eBioscience, USA), anti-BTLA (Abcam, USA) antibodies at 4 °C overnight. After washing with TBST (5 min, three times), sections were incubated with donkey anti-rabbit Alexa 488 and donkey anti-mouse Alexa 555 (Jackson ImmunoResearch, West Grove, PA, USA) in the dark for 30 min. Finally, sections were incubated with 1 μg/mL DAPI (Sigma, CA, USA) for 10 min to stain nuclei. The results were analyzed using an inverted Eclipse Ti-S microscope (Nikon, Japan). Liver sections were stained with hematoxylin & eosin (H&E).

Sections were air-dried and microwaved for 10 min in 10 mM citrate buffer. 10% donkey serum was used for blocking during staining. Sections were then incubated with diluted primary antibody as described below for 18 hours at 4 °C. Slides were washed 3 times in PBS for 5 min and then incubated with diluted secondary antibody for 1 h at room temperature. Slides were washed in PBS again and mounted in Vectashield medium with DAPI (Vector Laboratories). The primary antibodies used were rabbit anti-TFEB (1:250, Bethyl Laboratories), mouse anti-Ki-67 (1:1000, EBIOSCIENCE), rabbit anti-lysozyme (1:1000, DACO) and rabbit anti-apolipoprotein A1 Antibody (1 ug/ml, Invitrogen). The secondary antibodies used were donkey anti-Rabbit Alexa488 (1:200, Jackson Immunoresearch), donkey anti-Mouse Alexa488 (1:200, Jackson Immunoresearch), and donkey anti-Rabbit Alexa 594 (1:200, Jackson Immunoresearch). Images were collected using a confocal fluorescent microscope (Nikon, Melville, NY).

Embryos were isolated in PBS⁺ dissection medium [PBS containing Mg²⁺ and Ca²⁺]. Isolated embryos were fixed for 20 min at RT in 2% PFA in PBS⁺ followed by permeabilizing for 10-15 min in permeabilization solution [0.1 M glycine/0.1% Triton X-100]. Embryos were transferred into blocking solution [0.1% Tween-20; 10% FCS; 0.1% BSA; 3% Rabbit, Goat or Donkey serum]. Primary antibodies were added into the blocking solution and incubated o/n at 4 °C. The following antibodies were used: Foxa2 (Abcam, ab40874, 1:1000), Sox17 (Acris/Novus, GT15094,1:1000), GFP (Aves, GFP1020, 1:1000), AFP (R&D, AF5369, 1:1000) and IAP-GAG (Cullen lab, 1:1000). The next day, embryos were kept at RT for 2 hours. After 3 washes with PBST, embryos were incubated with secondary antibodies Donkey anti rabbit Alexa488 (Jackson Immuno Research, 711-545-152, 1:800), Donkey anti goat Alexa 555 (Invitrogen, A-21432, 1:1000), Donkey anti Chicken IgY Alexa488 (Jackson Immuno Research, 703-545-155, 1:800), Donkey anti mouse Cy3 (Jackson Immuno Research, 715-165-150, 1:800), Donkey anti rabbit Alexa647 (Jackson Immuno Research, 711-605-152, 1:500) for 3 hours at RT, followed by three washes. Embryos were then embedded in antifade medium (Invitrogen, P36930) for microscopy analysis.

Following primary antibodies were used for Immunofluorescence (IF) and Western blotting (WB): rabbit anti-GFP (1:200 IF, Invitrogen, A11122), chicken anti-GFP (1:200 IF, Aves, GFP-1020), rabbit anti-GFAP (1:100 IF, Dako, Z0334), mouse anti-NeuN (1:50 IF, Millipore, MAB377), rat anti-L1 (1:100 IF, Millipore, MAB5272), rabbit anti-Calretinin (1:500 IF, Millipore, MAB5054), goat anti-Nrp1 (2 µg/ml for function blocking, R and D, AF566), goat anti-VEGFR2 (1:15 IF, R and D, AF644), rabbit anti-VEGFR2 (1:1000 WB, Cell Signaling, 2479), rabbit anti-(phospho)VEGFR2 (Y1175) (1:500 WB, Cell Signaling, 2478), rabbit anti-(phospho/SFK (Y416) (1:200 IF, Invitrogen, 44660G), mouse anti-beta-III-tubulin (1:150 IF, Sigma, T5076), goat anti-VE-Cadherin (1:1000 WB, R and D, AF1002), Phalloidin (1:400 IF, Sigma, P1951). The following secondary antibodies were used: donkey anti-rabbit Alexa488 (Jackson Immunoresearch, 711-545-152), donkey anti-rabbit Alexa568 (Life Technologies, A10042), donkey anti-goat Alexa568 (Molecular Probes, A11057), goat anti-mouse Alexa568 (Invitrogen, A11031), goat anti-rat Alexa568 (Invitrogen, A11077), goat anti-chicken Alexa488 (Molecular Probes, A11039), donkey anti-goat HRP (Jackson Immunoresearch, 705–0350147), donkey anti-rabbit HRP (Jackson Immunoresearch, 711-035-152), donkey anti-mouse HRP (Jackson Immunoresearch, 715-035-150).