Alexa fluor 594 conjugated goat anti rabbit igg

To count parasites per vacuole in LEW rat BMDMs, coverslips containing 2 x 10⁵ LEW rat BMDMs were infected with 1 x 10⁵ parasites for 30 min followed by removing the uninvaded parasites by washing 3 times with PBS. After 24 h infection, the cells were fixed with 4% Paraformaldehyde (PFA) for 20 min followed by permeabilization/blocking with PBS containing 3% (w/v) BSA, 5% (v/v) goat serum (Thermo Scientific, Cat# 16210072), and 0.1% Triton X-100 for 30 min. The parasites were detected by incubating the coverslips with mouse anti-SAG1 DG52 (1:100 dilution) and rabbit anti-GRA7 (1:3000 dilution) antibodies for 1 h at room temperature. After incubating with secondary antibodies Alexa Fluor 488-conjugated goat anti-mouse IgG (1:3000 dilution) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:3000 dilution) together with DAPI (Sigma-Aldrich, Cat# D9542) at the final concentration of 1 μg/mL, the coverslips were mounted with Vecta-Shield mounting oil and the microscopy was performed with NIS-Elements software (Nikon) and a digital camera (CoolSNAP EZ; Roper Scientific) connected to an inverted fluorescence microscope (Eclipse Ti-S; Nikon). The number of parasites in at least 100 vacuoles was observed, counted, and quantified.

Nerve microtissues were fixed with 4% paraformaldehyde for 30 minutes and immunostained with rabbit anti-S100 antibody (1:200; Sigma-Aldrich) overnight at 4°C, followed by incubation with Alexa Fluor-488-conjugated goat anti-rabbit IgG (1:200; Abcam, Cambridge, UK) at room temperature for 2 hours. Finally, samples were mounted with 4′,6-diamidino-2-phenylindole (DAPI) and coverslipped. Images were taken on a fluorescence microscope (Olympus, Tokyo, Japan).
SCs were fixed with 4% paraformaldehyde for 30 minutes and immunostained with rabbit anti-S100 antibody (1:200) overnight at 4°C, followed by incubation with Alexa Fluor-594-conjugated goat anti-rabbit IgG (1:200; Abcam) at room temperature for 2 hours. Finally, samples were mounted with DAPI, coverslipped, and imaged by fluorescence microscopy.
DRGs were fixed with 4% paraformaldehyde for 30 minutes and immunostained with rabbit anti-S100 antibody (1:200) or mouse anti-neurofilament 200 antibody (1:400; Sigma-Aldrich) overnight at 4°C, followed by incubation with Alexa Fluor-488-conjugated goat anti-mouse IgG (1:200) or Alexa Fluor-594-conjugated goat anti-rabbit IgG (1:200) at room temperature for 2 hours. Finally, samples were mounted with DAPI and coverslipped. Images were obtained by fluorescence microscopy. Axon length was measured using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).