Alexa fluor 594 conjugated secondary antibody

The target cells were washed with cold PBS, fixed in methyl alcohol, and permeabilized with 0.5% TritonX‐100 (Sigma, USA) for 15 min. The cells were then washed with PBS three times and blocked with 4% BSA in PBS for 1 h at room temperature. Afterward, the cells were incubated with the primary antibodies against β‐catenin (Affinity, OH, USA, AF6266 or Abcam, Boston, USA; ab32572), YAP1 (Affinity, OH, USA, DF3182 or AF6328), ERα (Affinity, OH, USA, AF6058 or Abcam, Boston, USA; ab66102), ERβ (Affinity, OH, USA, AF6469), TEAD1 (Abcam, Boston, USA; ab133535), or LEF1 (Abcam, Boston, USA; ab137872) at 4°C overnight and subsequently incubated with normal goat serum (Beyotime Biotechnology, Shanghai, China) at room temperature for 30 min and subsequently with Alexa Fluor 594‐conjugated secondary antibodies (ProteinTech, Wuhan, China, SA00006‐4) and DAPI (Sigma, USA). The signal was visualized by confocal laser microscopy (FLUOVIEW FV1000; Olympus, Tokyo, Japan).

HSC-4 and SCC-9 cells (5 × 10⁴ cells per well) were seeded on 24-well plates and treated with NaB (2.5 or 5 mM) for 24 h. Then, the treated cells were fixed with 4% paraformaldehyde for 30 min at 4 °C. After that, cells were washed with PBS, permeabilized for 5 min in PBS containing 0.5% Triton X-100, and blocked for 1 h with PBS containing 1% BSA at room temperature. Subsequently, primary antibodies against E-cadherin, Vimentin, and SNAI1 (1:200 in PBS containing 1% BSA) were incubated overnight at 4 °C. After washing with PBS, cells were immunostained with Alexa Fluor 594-conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA) at 1:200 dilution protected from light at room temperature for 1 h. Finally, the immunostained cells were counterstained with DAPI (Beyotime Biotech. Co., Shanghai, China) and images were captured with a fluorescence microscope (Nikon DS-Ri2).

UL13-HEK293T cells or control cells were transfected with poly(I:C) for 3 h and then fixed for 20 min with 4% cold paraformaldehyde. Cells were then permeabilized for 10 min with 0.1% Triton X-100, and then blocked with 5% bovine serum albumin (BSA, Sigma) for 30 min. Cells were incubated with the appropriate primary antibodies for 120 min, followed by staining with Alexa Fluor 594-conjugated secondary antibodies (Proteintech Group Inc. China) or 647-conjugated secondary antibodies (TIANGEN, Beijing, China) for 60 min. Nuclei were stained with DAPI. Images were visualized and acquired using a laser scanning confocal microscope with LAS X software (Leica, Wetzlar, Germany).

Caco-2 cells were fixed using 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.2% Triton X-100 for 10 min, and then blocked with 2% bovine serum albumin in PBS at 37 °C for 30 min. The specimen slides were incubated with the primary anti-PKCζ or anti-TRAF2 antibody at 4 °C overnight, subsequently washed three times with PBS, and then incubated with FITC- (ZSGB-BIO) and Alexa Fluor 594-conjugated secondary antibodies (Proteintech) at 37 °C for 1 h. After additional washes with PBS, the specimen slides were counterstained with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) at room temperature for 10 min. An 80i Nikon microscope (Tokyo, Japan) was used to examine the immunofluorescent images.

For IHC, the liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4-μm thickness). They were then dewaxed, hydrated for 30 min at room temperature, and incubated overnight at 4 °C with the following primary antibodies: anti-CD31 (1:1000, Proteintech, Wuhan, China); anti-CD206 (1:10000, Proteintech, Wuhan, China), anti-HECTD3 (1:400, BIOS, Beijing, China). Subsequently, the slides were incubated with a horseradish peroxidase-labeled secondary antibody, and the immunoreactivity was visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB). Tissue sections were counterstained with hematoxylin and visualized using Leica Microsystems at 200 × and 400 × magnification. Image-pro plus 6.0 was used for image analysis (Media CybernetiHOPE, Inc., Rockville, MD, USA).
IF was performed as previously reported^{44 (link)}. Specifically, the sections or cell slides were incubated with anti-CD31, anti-CD206, anti-TRAF3 (1:100, Proteintech, Wuhan, China), anti-Ly6G (1:100, Novus, Littleton, USA), anti-MPO (1:200, Servicebio, Wuhan, China), anti-CD11b (1:500, Servicebio, Wuhan, China) and anti-HECTD3 (1:100, BIOS, Beijing, China) antibodies. After washing, the slides were incubated with Alexa Fluor 594-conjugated secondary antibodies or Alexa Fluor 488-conjugated secondary antibodies (1:200, Proteintech, Wuhan, China).