Alexa fluor 488 labeled goat anti rat secondary antibody

Fluorescent immunohistochemical analysis of PECAM-1 expression was performed using a Leica Bond Rx automated staining platform. Mouse tissues (kidney, liver, heart and testis) were embedded in Optimal Cutting Temperature (OCT) compound and snap frozen in isopentane-cooled liquid nitrogen. Samples (4 μm) were cryo-sectioned and placed onto charged slides. All sections were fixed in 4% paraformaldehyde for 20 min at room temperature and blocked with Background Sniper (Biocare Medical) for 30 min. The sections were stained with CD31 rat monoclonal antibody (Biocare Medical) at a dilution of 1:100 for 60 min at room temperature. Negative control tissues were incubated without primary antibody. The sections were subsequently incubated with an Alexa Fluor 488-labeled goat anti-rat secondary antibody (Thermo Fisher Scientific) at a dilution of 1:500 for 45 min at room temperature in the dark. Sections were washed and stained with DAPI (Sigma Aldrich) at 1:5000 dilution in DiH₂0 for 3 min. The stained tissues were visualized using a Nikon TI2-E inverted motorized microscope. Representative images are shown.

Mice were fed with 4% DSS received P-Tofa-Alexa 647 (i.v. 200 mg polymer/kg mouse, mixed with P-Tofa to a total Tofa equivalent dose of 210 mg/kg) on day 7 and sacrificed 1 day later. Colons were collected and prepared for histologic examination as described above. Tissue sections (20 μm) were immunohistochemically stained after antigen retrieval using citrate buffer with the following antibodies: rabbit anti-IBA1 (Wako, 019–19741, dilution 1:250), rat anti-CD326(EpCAM) (eBioscience, 14–5791-85, dilution 1:250) and rabbit anti-CD146 (Abcam, ab75769, dilution 1/250), respectively, overnight at 4 °C after antigen retrieval and 10% normal goat serum blocking. Slides incubated with rat anti-CD326 were further incubated with Alexa Fluor 488-labeled goat anti-rat secondary antibody (Thermo Fisher scientific, A11006, dilution 1:1000) and slides incubated with rabbit anti-IBA1 and rabbit anti-CD146 were incubated with Alexa Fluor 488-labled goat anti-rabbit secondary antibody (Thermo Fisher scientific, A11008, dilution 1:1000) for another 1 hr at 20 °C in the dark. The stained slides were imaged using a ZEISS LSM 800 confocal microscope after mounted in ProLong® Gold antifade mountant with DAPI (Thermo Fisher scientific, P36931).