Alexa fluor 488 f ab 2 goat anti rabbit igg

Ten microliters of cultured samples were placed on a glass slide with a 7mm well as previously described [48 (link)]. Briefly, slides were air dried overnight and fixed in acetone for 15 minutes and then placed in a humid chamber; 50μL of rabbit anti-LipL32 sera (1:250) was added to each spot and then incubated for 1 hour at 37°C. Next, slides were washed for 10 minutes in PBS with gentle rocking and incubated with secondary Alexa Fluor 488 F(ab’)2 goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) for 1 hour at 37°C in the dark. After extensive washing, slides were dried and counterstained for 15 seconds with Flazo Orange (1:50, National Veterinary Services Laboratory). Slides were then rinsed with PBS and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher, Waltham, MA, USA). Microscopic examination was done using a Nikon Eclipse E800 microscope and B2-A filter (excitation, 450–490 nm; emission, 520 nm) at 400× magnification [49 (link)].

After colony recovery from agar plates, 10 μL of the homogenized suspensions were placed on a glass slide with a 7 mm well for the fluorescent antibody test (FAT), as previously described²⁹ . In brief, slides were air dried overnight and fixed in acetone for 15 min. Slides were then placed in a humid chamber and 50 μL of rabbit anti-LipL32 sera (1:250) was applied to each 7 mm spot. Slides were incubated at 37 °C for 1 h and subsequently washed for 10 min in PBS with gentle rocking. Next, slides were incubated with secondary Alexa Fluor 488 F(ab’)2 goat anti-rabbit IgG (Invitrogen) for 1 h at 37 °C in the dark. After extensive washing, slides were dried and counterstained for 15 s with Flazo Orange (1:50, National Veterinary Services Laboratory). Slides were rinsed with PBS and mounted using ProLong Diamond Antifade Mountant with DAPI (Thermo-Fisher Scientific). Microscopic examination was done using a Nikon Eclipse E800 microscope and B2-A filter (excitation, 450–490 nm; emission, 520 nm) at 400 × magnification.