Alexa fluor 594 donkey anti goat igg

For immunofluorescent assay, the 143B, MG63, Saos-2 and Hs888 cells were fixed in a 4% paraformaldehyde solution for 5 minutes at 4° C and then cells were permeabilized with PBS + 0.01% Tween 20 for 20 minutes at room temperature. Subsequently, the cells were incubated with goat IgG polyclonal antibody anti-MLL3 (Q-20, Santa Cruz Biotechnology, Dallas, TX, USA) 1:50, at 4° C overnight. The following day, cells were washed with PBS buffer + 0.01% Tween 20 and subsequently cells were incubated with secondary antibody (Alexa Fluor 594 donkey anti-goat IgG; Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Then, the nuclei were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 10 minutes. Negative control was obtained by omitting the primary antibody. The fluorescence signal was examined with a fluorescence microscope Olympus IX50 with appropriate filters. Under 40X magnification, images of positive representative fields were captured.

Frozen 5 μm-thick tissue sections were fixed in cold acetone for 15 min, air-dried, and treated for 30 min with a blocking reagent [10% fetal bovine serum (FBS) in PBS]. After washing with PBS, the sections were incubated with primary antibodies overnight at 4°C, and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) for anti-NP and anti-pSFK (Tyr416) antibodies, Alexa Fluor 594 donkey anti-goat IgG (Invitrogen) for anti-MGL1/2 antibody, Texas Red-X goat anti-rat IgG (Invitrogen) for anti-CD3 antibody, and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen) for anti-MPO antibody for 2 hrs at room temperature (RT). The sections were mounted with CC/Mount (Diagnostic BioSystems, Pleasanton, CA) containing DAPI (Dojindo Laboratories, Kumamoto, Japan). Fluorescent images were visualized using BIOREVO BZ-9000 (Keyence, Osaka, Japan).

Blood was collected in heparin-containing vials when mice were killed, centrifuged at 4°C (3,000 rpm in 20 min), and stored at −80°C. Plasma Gal-3 levels were detected by a mice Galectin-3 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) in twin duplicates wells, following protocols provided by the manufacturer. The detection range of the plasma Gal-3 was 8.19–2,000 pg/mL, and the limit of detection was 6 pg/mL.
Paraffin-embedded LV sections (6 μm) were prepared and used for Gal-3 immunofluorescent staining within mice. For Gal-3 immunofluorescent staining, after dewaxed, heat-induced antigen retrieval and permeabilization were carried out (with 10 mM of Na-citrate buffer containing 0.05% Tween 20; pH 6.0; 95°C for 25 min) followed by blocking with DAKO Protein Block (X0909, Agilent, 1 h at room temperature). Sections were incubated with primary goat anti-mouse Gal-3 (1:100, AF1197, R&D Systems) overnight at 4°C, then they were incubated with the secondary antibody, Alexa Fluor 594 donkey anti-goat IgG (1:200, A11058, Invitrogen by Thermo Fisher Scientific). The cardiomyocyte boundary was revealed by wheat-germ-agglutinin FITC staining (1:80, FL-1021, Vector Labs, 1 h at room temperature). Images were acquired with an Olympus BX61 fluorescent microscope.

For cell culture, primary and secondary antibodies were used at 1:300 concentrations. EGFR clone 225 (Milipore). MUC-1 Ab-5 (Thermo Fisher Scientific). EEA1 H-300 (Santa Cruz Biotechnology). COX-IV (Cell Signaling 3E11). TGN46 (Sigma 7576). FAK A-17 (Santa Cruz) was used at a 1:50 primary and 1:100 secondary concentration. For mammary glands, primary antibodies were used at 1:300 concentrations; secondary antibodies were used at 1:500 concentrations. EGFR 1005-G (Santa Cruz Biotechnology). AlexaFluor 488 donkey anti-mouse IgG (Invitrogen), AlexaFluor 594 donkey anti-rabbit IgG (Invitrogen), AlexaFluor 594 donkey anti-goat IgG (Invitrogen), AlexaFluor 647 goat anti-armenian hamster IgG (Jackson ImmunoResearch).

Pancreatic specimens from KC mice were snap frozen in liquid nitrogen and 8 uM-thick sections were cut using a cryotome. The samples were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton-X-100. Samples were blocked in normal donkey serum then incubated with 1:100 dilution of the primary antibodies Goat Anti-Amylase C-20 and sc-12821 Rabbit Anti-CK-19 H-60 (Santa Cruz Biotechnology Inc.) overnight at 4^°C. Samples were then incubated with the secondary antibodies Alexa fluor 488 Donkey anti-rabbit IgG (Invitrogen A-21206) or Alexa-fluor 594 Donkey anti-goat IgG (Invitrogen A-11058) at a 1:500 dilution for 1 hour at room temperature, then mounted with ProLong Gold antifade reagent with DAPI (Life Technologies P36931). Total number of lesions with cells co-staining for amylase and CK-19 were quantified per high power field in merged images from a Zeiss Axio Imager widefield fluorescence microscope in a blinded fashion.