Goat anti rabbit cy2

Cryosections of 3.7% neutral-buffered formalin-fixed tumors were stained with the following antibodies: CD45 rat anti mouse 1:50 (BD Pharmingen; clone: 30F11); CD11b rat anti mouse 1:50 (BD Pharmingen; clone: M1/70); and F4/80 rat anti mouse 1:50 (eBioscience; clone: BM8). The secondary antibody used was goat anti rat Alexa 594 1:500 (Dianova #112-585-062). Active caspase 3 rabbit anti mouse 1:100 (Abcam, #ab2303) combined with goat anti rabbit Cy2 1:500 (Dianova, #111-227-003) was used to determine apoptosis in tumors.
To determine the degree of liver fibrosis, 5 μm frozen sections were stained. Sections were fixed in 4% paraformaldehyde. Extracellular matrix was stained using picrosirius red. Collagen type I was stained using rabbit anti collagen-type-I (Millipore, #AB765P); Cre-recombinase was stained using rabbit anti cre (Novagen, #69050–3) and goat anti-rabbit conjugated with Cy2 (Dianova, #111-227-003).
Sections were photographed using a Keyence Biozero microscope (Keyence, Germany) and processed using ImageJ. Quantification was performed in at least three sections per mouse in at least three mice per group or more, as noted in the figure legends. At least 0.9 mm² was examined per section.

Confluent HFF were grown on glass coverslips in 24-well plates and were infected with N. caninum tachyzoites, either NC-1 isolate or different clones expressing variants of hem-agglutinin-tagged NcGRA9 (NcGRA9-HA variants). Infected cultures were maintained for different time spans, depending on the experiments as indicated in the text. The immunofluorescent staining was performed according to Guinoaud et al. (2010) with the following adaptations: methanol/acetone permeabilization for 20 min, final DAPI concentration 0.5 μg/ml, coverslips mounted in Fluoromount-G (Southern Biotech, Birmingham, USA), and analyses with Zeiss LSM 780 confocal microscope.
For immunolabeling of extracellular N. caninum tachyzoites the parasites were harvested as described above, suspended in medium and small droplets of the suspension was placed onto coverslips. The samples were air-dried and immunofluorescence staining was performed as described above.
Primary antibodies used were rabbit anti-NcGRA9 antiserum or rabbit anti-HA (Invitrogen, Carlsbad, USA), both used at a dilution of 1 : 1000. As secondary antibodies goat anti-rabbit cy2 or cy3 (Dianova, West Grove, USA) were used at 1 : 1000 dilution.