Alexa fluor 568 goat anti mouse igg

Paraffin sections were treated with xylene for 10 min three times. The sections were hydrated through a graded alcohol series and then rinsed three times with distilled water. After the sections were blocked for 20 min in 10% goat serum in PBS, they were incubated overnight at 4°C with the PPARα-specific rabbit polyclonal antibody (1:1000; ab8934, Abcam) and the CHOP-specific mouse monoclonal antibody (1:1000; 2895, Cell Signaling Technology Inc.). The slides were then incubated with Alexa Fluor^® 488 goat anti-rabbit IgG or Alexa Fluor^® 568 goat anti-mouse IgG (1:200; A-11034 and A-11031, respectively, Invitrogen) for 45 min. After three washes with PBS, the nuclei were stained with DAPI (1 μg/ml; Shizebio) for 10 min. The images were examined on a Nikon Eclipse E800 fluorescent microscope.

Immunofluorescence staining was performed as described before²² . Fixed hBMSC were incubated with goat anti-hMMP2-IgG (BioTechne) or mouse anti-hTIMP3-IgG (BioTechne) followed by incubation with AlexaFluor488 rabbit anti-goat-IgG (Invitrogen, Karlsruhe, Germany) or AlexaFluor568 goat anti-mouse-IgG (Invitrogen). Nuclei were stained with 0.2 µg 4′,6-diamidino-2-phenylindole (DAPI)/mL and finally samples were embedded in Mowiol 4–88 (Sigma). An Olympus IX70 microscope (Carl Zeiss, Oberkochen, Germany) was used for visualization. Digital images were obtained with an AxioCam MRm camera (Carl Zeiss) using AxioVision software release 4.9 (Carl Zeiss).
For visualization of colocalization of SH/MMP2/TIMP3, hBMSC were incubated with ATTO655-labeled SH (200 µg/mL).
Colocalization analysis of immunofluorescence signals was performed as previously described²² . The immunofluorescence signals of ATTO655-SH and MMP2 respectively TIMP3 or MMP2 and TIMP3 were quantified with software Fiji setting threshold automatically by Otsu filter. Colocalization values (Pearson, Manders’ coefficients) were calculated from eight representative images using the colocalization plugin JACoP (Just another colocalization plugin)^{51 (link)}.

Stomach biopsy specimens (antrum and stomach body) of H. pylori-positive patients were fixed with formalin, embedded in paraffin, and sliced into 4-μm-thick sections. Tissue sections were deparaffinized and antigen retrieval was performed via heat treatment with a Target Retrieval Solution (DAKO North America, Inc., Carponteria, CA, USA) diluted with distilled water. Thereafter, sections were incubated with Protein Block Serum Free solution (DAKO North America, Inc.) at room temperature for 20 min. An anti-H. pylori rabbit polyclonal antibody (DAKO North America, Inc.; 1:150) and a mouse monoclonal anti-human GGT antibody (Abnova, Taipei, Taiwan; 1:150) were applied as primary antibodies and incubated with the tissue sections in a humidified chamber overnight at 4 °C. The slide was washed thrice in PBS with Tween-20 (PBS-T) for 5 min each. Thereafter, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA; 1:200) and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen; 1:200) were applied as secondary antibodies and incubated with the tissue sections for 1 h at room temperature in the dark. The slide was washed thrice in PBS-T for 5 min each. Fluorescence microscopic images were obtained using an All-in-One Fluorescence Microscope (BZ-X700; KEYENCE Japan, Osaka, Japan), and images were captured at × 1000 magnification.

Frozen sections or cells on slides were fixed in paraformaldehyde for 10 min, washed in PBS buffer three times, and subsequently treated with Triton X-100 to permeabilize cell membranes. The sections were blocked in 10% goat serum and 3% BSA in PBS for 20 min. The following antibodies were incubated with the sections overnight at 4 °C: PPARα (1:500, Abcam), CHOP and LC3 (1:500, Cell Signaling Technology). After washing in PBS, the slides were incubated with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 568 goat anti-mouse IgG (1:250, Invitrogen, ThermoFisher Scientific, Inc.) for 30 min at room temperature. The nuclei were stained with DAPI (1 µg/ml, Abcam) for 5 min. Finally, the images were analyzed by a Leica DM2500 fluorescence microscope.

Cells were cultured overnight on gelatin precoated coverslips and fixed with paraformaldehyde 4% for 10 min in room temperature. After incubation with block (PBS 5% BSA, 0.5% triton) for 30 min, coverslips covered with monoclonal mouse anti-CREM (clone 3B; Novusbio) diluted 1:100 in block, for 1–2 h, in room temperature. The coverslips were washed three times with PBS incubated with the Alexa Fluor 568 goat anti-mouse IgG (1:500, Invitrogen; Waltham, MA, USA). For visualization of actin filaments the cells were incubated with Phalloidin Alexa 488 diluted 1:500 in block for 1 h, at room temperature. The mounting medium contained DAPI for staining nuclei (ProLong® Gold Antifade Mountant with DAPI, Life Technologies; Carlsbad, CA, USA). After each staining step, the cells were washed three times with PBS. Images were taken with a Nikon Elipse Ni fluorescence microscope (Nicon; Tokio, Japan) and analysed with ImageJ v1.53f51 software (http://rsbweb.nih.gov/ij/).

To assess the extent of Burkholderia escape from phagosomes of activated (50 U/mL IFN-γ) Nramp1⁺ or Nramp1⁻ macrophages, we examined co-localization of bacteria with lysosome-associated membrane protein 1 (LAMP1) at 4 h p.i. The bacteria were stained red with mouse anti-Burkholderia monoclonal antibody (9D5; 1:20; from the laboratory of Dr. Narisara Chantratita) and Alexa Fluor 568-goat anti-mouse IgG (1:500; Invitrogen, USA). LAMP1 was stained green with rat monoclonal antibody (1D4B; 1:200; Abcam, USA) and Alexa Fluor 488 goat anti-rat IgG antibody (1:500; Molecular Probes, USA), and nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI; 1:500). All of the staining procedures were performed for 1 h at 37°C. Cells were examined by a laser-scanning confocal microscope equipped with LSM5 Image Browser (LSM 510 META, Carl Zeiss, Germany). The percentage of intracellular Burkholderia associated with LAMP1 was determined as the number of bacteria co-localized with LAMP1/total number of bacteria within macrophages × 100. The association of Burkholderia with LAMP1 was considered when the red fluorescent bacteria co-localized with the green fluorescence of LAMP1-positive vacuoles, represented as an area of yellow staining.