Alexa fluor 488 goat anti rat igg h l antibody

Staining of tumor sections was done as previously described.9 Briefly, tumor samples excised from BALB/c nude mice were snap‐frozen in a dry‐ice acetone bath. Frozen samples were further sectioned at 10‐μm thickness in a cryostat and subsequently incubated with primary mAbs to platelet and endothelial cell adhesion molecule 1 (PECAM1) (Mec13.3) (BD Pharmingen, Franklin Lakes, NJ, USA), followed by incubation with secondary Alexa Fluor 488 goat anti‐rat IgG (H+L) antibody (Invitrogen). Specimens were then examined using an LSM 510 META confocal microscope (Carl Zeiss, Feldbach, Switzerland). All images were imported into Adobe Photoshop as JPEGs or TIFFs for figure assembly. Images were processed using ImageJ (NIH, https://imagej.nih.gov/ij/) to quantify PECAM1‐positive areas.

In the footpad spontaneous metastasis model, the popliteal lymph node was resected, fixed with 4% (w/v) PFA overnight at 4°C, and embedded in paraffin. Five-micron sections were deparaffinized and subjected to HE staining and immunofluorescence. For immunofluorescence, anti-CEA antibody (1:200 dilution, #10094, IBL) and anti-GFP antibody (1:1000 dilution, #04404-26, anti-GFP [Rat IgG2a] monoclonal [GF090R]; Nacalai Tesque, Tokyo, Japan) were used as primary antibodies. The sections were then washed three times in Tris-buffered saline with Tween 20 (TBS-T; 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20), followed by incubation at room temperature for 3 h with Alexa Fluor 568-conjugated anti-mouse IgG (1:500 dilution, #A-11004, Alexa Fluor 568 Goat Anti-Mouse IgG [H+L] Antibody; Invitrogen) and Alexa Fluor 488-conjugated anti-mouse IgG (1:500 dilution, #A-11006, Alexa Fluor 488 Goat Anti-Rat IgG (H+L) Antibody; Invitrogen) as the secondary antibodies. Fluorescence and differential interference contrast images of the sections were observed with a wide-field fluorescence microscope (PlanApo 20×/0.75, PlanApo 40×/0.95, ECLIPSE 80i; Nikon).