Donkey anti rabbit igg alexa 594

Cells were fixed at days 9, 11, 14, 16, 18, 21, 23, and 25 by incubation in 4% formaldehyde (Cat. No. 02176, Histolab) for 10 min at RT. The cells were permeabilized by incubation in 1% Triton X-100 (Cat.No. T8787, Sigma-Aldrich) in D-PBS +/+ (Cat. No. 14040-091, Gibco) for 15 min at RT, then the cells were blocked in 2% BSA (Cat. No. A9418, Sigma-Aldrich) in D-PBS +/+ for 60 min at RT. The cells were immunostained for the markers CK18 (Cat. No. MA5-12104, Invitrogen) and CAR (Cat. No. MA5-29208, Invitrogen). The antibodies were diluted in 0.1% BSA in D-PBS +/+ (1:100 for primary antibodies, and 1:1000 for secondary antibodies). The primary antibodies were incubated overnight at 4 °C. The secondary antibodies Goat anti-mouse IgG Alexa 488 (Cat. No. A11029, ThermoFisher), Donkey anti-rabbit IgG Alexa 594 (Cat. No. A21207, ThermoFisher), and DAPI were incubated in the dark for 2 h at RT. The photographs were processed by applying ImageJ software (http://imagej.nih.gov).

The received tumor tissue and acoustic cell clusters were fixed in 4% paraformaldehyde followed by dehydration, paraffin embedding, sectioning, and standard H&E staining. Immunofluorescence was performed according to a previously published protocol. Briefly, POCCs were washed in PBS twice and blocked in 500 μL of blocking buffer for 60 min. The primary antibodies for EpCAM, CD3, and SMA were diluted at the ratio of 1:200 in the antibody dilution buffer. At the end of the blocking procedure, the blocking solution was aspirated completely. Acoustic cell clusters were incubated with primary antibodies at 4 °C overnight and washed 3 times with PBS solution. Secondary antibodies, Donkey anti-Rabbit IgG-Alexa 594 (Thermo Fisher, R37119), Donkey anti-Rat IgG-Alexa 488 (Thermo Fisher, A-21208), were diluted at a ratio of 1:500 in the antibody dilution buffer and samples were incubated with secondary antibodies for 2 h at room temperature in the dark. Then the samples were rinsed three times in PBS solution for 5 min each time. The acoustic cell clusters were immersed in a fructose-glycerol clearing solution and covered with coverslips. Stained samples were imaged using an X83 microscope.

HEK293 or HeLa cells were fixed with 4% paraformaldehyde and then permeabilized by treatment with PBS and 0.2% Triton-X100. The cells were blocked with 1% albumin in PBS and then incubated with primary antibodies. Subsequently, the cells were treated with one of the following appropriate secondary antibodies: Alexa 488 donkey anti-rabbit IgG, Alexa 594 donkey anti-rabbit IgG, Alexa 594 donkey anti-mouse IgG, Alexa Fluor 488 phalloidin, or Alexa Fluor 568 phalloidin (Molecular Probes). Cellular DNA was counterstained with DAPI (Dojindo). Images were captured using an Axioskop2 plus (Zeiss) fluorescent microscope equipped with Axiovision software.

For fluorescence staining, cell samples were fixed in 4% paraformaldehyde for 30 min at room temperature and washed three times with PBS (3 min each). The cells were then permeabilized and blocked with 2% normal donkey serum in 0.01 M PBS containing 0.3% Triton X‐100, 0.02% sodium azide and 0.12% carrageenan (pH 7.4, all reagents were purchased from Sigma‐Aldrich, St. Louis, MO, USA) for 1 hr at room temperature. Samples were subsequently incubated overnight at 4°C with the primary antibody. For double immunofluorescence, samples were incubated with a mixture of two primary antibodies followed by a mixture of the two respective secondary antibodies, Alexa 488 donkey anti‐mouse IgG (1:500, A21202, Invitrogen) or Alexa 594 donkey anti‐rabbit IgG (1:500, A21207, Invitrogen), and counterstained with DAPI (4′,6‐diamidino‐2‐phenylindole, 1:5000, Invitrogen). The following primary antibodies were used: mouse anti‐TNF‐α IgG (1:300, sc‐52746, Santa Cruz Biotechnology, San Diego, CA, USA), mouse anti‐IL‐10 IgG (1:300, sc‐57244, Santa Cruz Biotechnology) and rabbit anti‐β‐actin IgG (CW0096A, ComWin Biotech, Beijing, China; 1:2000). Confocal images were obtained using a confocal laser microscope (FV1000; Olympus, Tokyo, Japan), and digital images were captured with FluoView 1000 (Olympus).