Alexa fluor 594 conjugated goat anti rat igg

Immunohistochemistry was performed as described previously [6 (link), 18 (link)]. After anesthetization, all mice were perfused with 4% paraformaldehyde containing 0.5% picric acid. The brains were removed, fixed overnight, transferred to 30% sucrose, and stored at 4 °C. Coronal sections (14 μm) were generated using a cryostat. Consecutive sections were boiled in citrate buffer solution for 5 min and incubated with 2 N HCl at 37 °C for 30 min, followed by incubation in a blocking solution. The sections were incubated overnight with a monoclonal rat anti-BrdU primary antibody (1:5000; Novus Biologicals, Littleton, CO) and a monoclonal mouse anti-NeuN primary antibody (1:500; Millipore, Hayward, CA) in the blocking solution. Subsequently, the sections were incubated for 2 h with Alexa Fluor 594-conjugated goat anti-rat IgG (1:500; Invitrogen, Grand Island, NY) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; Invitrogen).

Commercial antibodies were BD34 (product number 610947; BD Biosciences, San Jose, CA); 2B1 (MA1-82452; ThermoFisher Scientific, Rockford, IL); mouse IgG1 isotype control (554121; BD Biosciences); anti-GFP (product number A6455; Invitrogen); anti-Flag (F7425; Invitrogen); Alexa Fluor 488-conjugated goat anti-rat IgG and goat anti-rabbit IgG (A21210 and A11034, respectively; Invitrogen); Alexa Fluor 594-conjugated goat anti-rat IgG (A21471, Invitrogen); horseradish peroxidase-conjugated goat anti-mouse IgG, anti-rabbit IgG, and anti-rat IgG (7076, 7074, and 7077, respectively; Cell Signaling Technology, Danvers, MA); and anti-6X-His tag (372900, Invitrogen) M2 Flag affinity resin was purchased from MilliporeSigma (Burlington, MA). TCEP, Lipofectamine 2000, and protein A/G agarose resin were obtained from ThermoFisher Scientific.

Vessel density in P29mtP29 and P29mtB82M tumours was determined by staining tissue sections for CD31. For this purpose, surgically removed tumours were immediately embedded and frozen in optimal cutting temperature (OCT) compound. Next, cryostat sections (8-µm thick) were cut and fixed in 4% paraformaldehyde for 10 min, blocked with 1% bovine serum albumin (BSA) in Dulbecco’s phosphate-buffered saline (DPBS) and then incubated with rat anti-mouse monoclonal CD31 antibody (BD Biosciences, 550274, 1:100). After being washed with DPBS, the sections were incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (Invitrogen, Thermo Fisher Scientific, A-11007, 1:300) for 1 h. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), the sections were observed under a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan). The pixel values of the CD31-positive areas were calculated for each image to determine the tumour vessel density using the ImageJ software (National Institutes of Health).

To identify the macrophages by immunohistochemistry, the sections were covered by blocking buffer (5% BSA) for 30 min at room temperature followed by primary and secondary antibody incubation for 1 h each at room temperature. Macrophages were identified by immunofluorescence staining with rat anti-mouse F4/80 monoclonal antibody (CI: A3-1, Bio-Rad) followed by Alexa Fluor^® 594 conjugated goat anti-rat IgG (Invitrogen, CA, United States). M1 pro-inflammatory macrophages were further identified by rabbit anti-mouse inducible nitric oxide synthase (iNOS) polyclonal antibody (Abcam, Cambridge, MA, United States) followed by Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Invitrogen, CA, United States). M2 anti-inflammatory macrophages were identified by rabbit anti-mouse liver Arginase (Arg1) polyclonal antibody (Abcam, Cambridge, MA, United States) followed by Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Invitrogen, CA, United States). ProLong Gold Antifade Mount with DAPI (Life Technologies, Grand Island, NY, United States) was used to mount the slides. A fluorescence microscope (Digital Microscope, Keyence, IL, United States) was used to detect the immunohistochemistry staining. Finally, the cells were manually counted in 3 randomly selected fields of view by Image J software (National Institutes of Health, United States).