Alexa fluor 647 donkey anti rat igg

Tumor tissues were fixed in 4% paraformaldehyde for 3 hours, followed by incubation in 30% sucrose overnight at 4°C. The tissues were OCT embedded and kept at -80°C. Staining for the endothelial cell marker CD31 (1 : 200, clone MEC13.3, catalog 550274, BD Biosciences) was performed on frozen sections (20 μm thickness), followed by staining with secondary antibody Alexa Fluor 647 donkey anti-rat IgG (1 : 200, catalog 712-605-153, Jackson ImmunoResearch) in dark, humid chambers [27 (link)]. The slides were counterstained for cell nuclei with Sytox Green. Fluorescence images were obtained with an Olympus FV3000 confocal laser-scanning microscope. Microvessel density was assessed by using Image-Pro plus software (version 6.0). The variables were determined for 4-5 photographic areas from each tumor (640 × 640 μm² each). Confocal images were taken in randomly selected fields, excluding the tumor periphery.

The eyes for immunostaining were enucleated 7 or 14 days after photocoagulation and then fixed in 4% paraformaldehyde for 12 h. The RPE-choroid-sclera complex was separated from the retina, isolated, and blocked in non-immune horse serum (Vector Labs) for 1 h, and then incubated with the primary antibody at 4 °C overnight. Then, the RPE-choroid-sclera complex was stained with a secondary antibody for 1 h, and flat-mounted on the slide. The slides were viewed and photographed for an overall picture and FLUOVIEW FV10i (Olympus) for the laser spots. The intensity of CD68 and VEGF-A in the CNV lesion was determined by the ImageJ analysis software (National Institutes of Health). The accumulation of Iba-1⁺ myeloid cells around the CNV was counted in Iba-1-stained whole RPE-choroidal flat mounts viewed from the RPE side. The primary antibodies used were as follows: rabbit anti-Iba1 (1:200; FUJIFILM Wako, catalog 019-19741), sheep anti-mPGRN (1:200; R&D systems, catalog AF2557), rabbit anti-VEGF-A (1:200; Merck Millipore, catalog PC315), rat anti-CD68 (1:200; Bio-Rad, catalog MCA1957GA), Alexa Fluor^® 647 donkey anti-sheep IgG (1:1000; Invitrogen, catalog A21448), Alexa Fluor^® 647 donkey anti-rat IgG (1:1000; Jackson ImmunoResearch, catalog 712-605-153), and Alexa Fluor^® 546 donkey anti-rabbit IgG (1:1000; Invitrogen, catalog A10040).

Dissected human or mouse liver tissues were fixed in 4% paraformaldehyde at 4 °C for at least 12 h and incubated in 30% sucrose at 4 °C overnight. Samples were embedded in the optimum cutting temperature (OCT) compound and cut into 5-μm sections. After blocking with TBST containing 20% FBS, sections were incubated with anti-FXYD1 (Abcam, ab76597, 1:500), anti-GJB1 (Abcam, ab66613, 1:500), anti-HNF4A (Santa Cruz Biotechnology, sc-6556, 1:50), anti-SOX9 (Millipore, ab5535, 1:200), anti-FGB (Abcam, ab118533, 1:500), anti-ID3 (Abcam, ab41834, 1:500), and anti-VEGFR3 (Invitrogen, 14-5988-82, 1:100) antibodies at 4 °C overnight. After washing, sections were treated with Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, A11055, 1:1000), Alexa Fluor 594 donkey anti-goat IgG (Invitrogen, A11058, 1:1000), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, A11008, 1:1000), Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen, A21207, 1:1000), Alexa Fluor 647 donkey anti-rat IgG (Jackson ImmunoResearch, 712-605-150, 1:250), and Alexa Fluor 488 donkey anti-sheep IgG (Invitrogen, A11015, 1:1000). DAPI (Sigma, D9564, 0.5 µg/mL) was used for nuclear staining. Images were acquired using an LSM 710 NLO and DuoScan System (Zeiss).