Alexa fluor 546 conjugated goat anti rabbit

Immunofluorescence staining was performed as described previously.^{35 (link), 36 (link)} Mice were perfused intracardially with 4% paraformaldehyde and postfixed overnight in 4% paraformaldehyde at 4 °C, cryoprotected in 30% sucrose and embedded in OCT. Tissues were cryosectioned at 15 μm and mounted on slides before incubation with primary antibodies (SENP1, 1:100, IMG, Littleton, CO, USA; Iba1, 1:100, Abcam; S100β 1:100, Sigma, St. Louis, MO, USA; cleaved caspase-3, 1:100, CST, Danvers, MA, USA) overnight at 4 °C and then with Alexa Fluor546-conjugated goat anti-rabbit or Alexa Fluor488-conjugated goat anti-mouse antibodies (Invitrogen; 1:200) for 1 h at room temperature. Before incubating with primary antibodies, sections were rinsed in 0.1 M PBS (pH 7.4) with 0.2% Triton X-100 and blocked in goat serum for 1 h at room temperature. Sections were analyzed by epifluorescence microscopy (OLYMPUS IX71, Shinjuku-ku, Tokyo, Japan) or confocal fluorescence microscopy (Zeiss, Oberkochen, Germany, LSM 510).

Tax1BP1^+/+ mice were sacrificed and the liver tissue was fixed with 4% paraformaldehyde for 24 h. Ten micrometer-cryosections were prepared, rehydrated in PBS and blocked with 2% bovine serum albumin (BSA)/PBS solution containing 0.1% Triton X-100. The tissue was incubated with rabbit anti-TRAF6BP (= Tax1BP1; 1:100 dilution; Abcam) and with rat anti-mouse CD68 (1:100 dilution; Biolegend) in PBS containing 2% BSA and 0.1% Triton X-100 overnight at 4 °C. After washing with PBS, the sections were further incubated with Alexa Fluor 546 conjugated goat anti-rabbit (1:200 dilution; Invitrogen) and Alexa Fluor 633 conjugated goat anti-rat (1:200 dilution; Invitrogen) for 1 h at room temperature. Both secondary antibodies were diluted in PBS containing 2% BSA and 0.1% Triton X-100. Sections without rabbit anti-TRAF6BP staining were used as negative controls. All coverslips were mounted on slides with Permount toluene solution (Fisher Chemicals) and imaged using an Olympus Fluoview 1000 confocal microscope.

Formalin fixed and paraffin-embedded (FFPE) peritoneal tissues were stained with mouse anti-GFAP A488-labeled (Biolegend, # 644704, 1:250), rabbit anti-tyrosine hydroxylase (abcam, #ab112, 1:1000), goat anti-NF-M (SantaCruz, #sc-16143, 1:50), rabbit anti-RGM-A (Abcam, #ab26287 1:50) or goat anti-RGM-A (SantaCruz, # sc-46481, 1:50) and IgG isotype control antibodies (Santa Cruz Biotechnology) as negative controls. Alexa Fluor 594-conjugated donkey anti-goat (Invitrogen, #A-11058, 1:100), Alexa Fluor 546-conjugated goat anti-rabbit (Invitrogen, #A-11010, 1:100) and Alexa Fluor 647-conjugated goat anti-rabbit (Invitrogen, #A-21244, 1:100) were used as secondary antibodies. Immunofluorescent pictures were acquired using a confocal microscope (LSM 510 Meta fluorescence microscope, Carl Zeiss) and ZEN software (Carl Zeiss). To perform immunohistochemical staining for PCNA, FFPE peritoneal tissues were stained with an anti-PCNA antibody (Santa Cruz Biotechnology) using a Vectastain ABC Kit (Vector Labs) and DAB peroxidase substrate (Sigma-Aldrich) according to the manufacturers’ instructions. As secondary antibody a biotin-conjugated rabbit-anti-mouse antibody (Jackson ImmunoResearch) was used. The sections were then counterstained with hematoxylin.

Eight mice were used for immunostaining experiments; the 3-month-old and 18-month-old groups included 2 male and 2 female mice each. Mice were perfused intracardially with 4% paraformaldehyde in PBS. After an overnight post-fixation in the same fixative at 4 °C, brain tissues were cut into 50 μm sections with a microtome. Brain sections were blocked with 0.3% Triton X-100 and donkey serum in PBS for 1 h at room temperature and then incubated with H3K9me3 (1:500, Abcam, ab 8898) or Lamin B1 (1:500, Abcam, Cat#229025) or L1-ORF-1p (1:200, Abcam, ab 216324) and CaMKIIα (1:300, ThermoFisher Scientific, MA1–048) primary antibody overnight at 4 °C. Next, brain sections were incubated with Alexa Fluor546-conjugated goat anti-rabbit (Invitrogen, 1:500, A-11035) or Alexa Fluor488-conjugated goat anti-mouse secondary antibodies (Invitrogen, 1:500, A-11029) for 1 h at room temperature, washed in PBS, and mounted in Vectashield containing DAPI (Vector Labs Cat#H-1500).

The captured tumor cells on the membrane were processed with Cytofix/Cytoperm Fixation/Permeabilization solution (BD, New Jersey, USA) for 10–15 min, incubated with 10% Goat Serum (Jackson, West Grove, USA) for 30 min at room temperature, then incubated with anti-CK8/18/19, anti-vimentin (Abcam Trading (Shanghai) Company Ltd., Shanghai, China) and anti-CD45 (Santa, Texas, USA) antibody overnight at 4 °C. The next day they were incubated with secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse, Alexa Fluor 546-conjugated goat anti-rabbit, Cy5-conjugated goat anti-rabbit (InvitrogenTM, Thermo Fisher Scientific, Waltham, USA), and Hoechst (SIGMA, St. Louis, MO) for 1 h at room temperature. Then they were imaged by fluorescence microscope.

Cells were fixed with 4% paraformaldehyde (Wako) for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 10% goat serum (Nichirei Bioscience) in PBS for 1 hr at room temperature (RT). Cells were incubated with primary antibodies for 2 hr at RT before incubation with appropriate secondary antibodies for 1 hr at RT, prior to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI)(Dojindo Laboratories). Samples were observed using a Leica fluorescent microscope (Leica Microsystems). The primary antibodies used were: anti-nestin antibody (1:100; Millipore), anti-fibronectin antibody (1:100; Sigma-Aldrich), anti-αSMA antibody (1:100; Sigma-Aldrich), anti-S100β antibody (1:100; Sigma-Aldrich) and anti-trichohyalin antibody (1:5; Santa Cruz Biotechnology). Secondary antibodies used were: Alexa Fluor 488-conjugated goat anti-mouse (1:200) and Alexa Fluor 546-conjugated goat anti-rabbit (1:200, both from Life Technologies).

Cells overexpressing ERroGFP-iLs were fixed with 4% paraformaldehyde for 20 min at room temperature, washed with phosphate buffered saline without calcium and magnesium (PBS (-)) containing 0.2% Triton X-100, and incubated and permeabilized with PBS (-) containing 1% glycerol, 1% bovine serum albumin, 1% normal goat serum and 0.2% Triton X-100 for 1 h. The fixed and permeabilized cells were incubated with rabbit anti-GFP antibody for 2 h, mouse anti-Hsp47 for 1 h, and then with Alexa Fluor 546-conjugated goat anti-rabbit and Alexa Fluor 633-conjugated goat anti-mouse (Life Technology) as the secondary antibodies for 1 h. ERroGFP-iLs in the fixed cells were excited using a 488 nm laser and fluorescence was detected using a 500–530 nm filter. Multicolour confocal fluorescence images were obtained by sequential scanning using an LSM 510 META confocal microscope (Carl Zeiss).