Immunofluorescence Microscopy of M. hyopneumoniae

Microscopy was performed following the same protocol as in [4 (link)]. Briefly, 1 ml of M. hyopneumoniae strain J culture was centrifuged at 10 000×g for 10 min and washed three times with 1 ml sterile PBS. A 1 in 100 dilution of cells was made in PBS and added to glass coverslips and allowed to settle for 15 min at room temperature. Paraformaldehyde (4%) was added and incubated at room temperature for 30 min. Non-specific binding sites were blocked using 2% BSA in PBS overnight at 4°C. Cells were incubated with either a 1 in 100 dilution of rMHJ_0461 antisera or control rabbit sera for 1 h at room temperature, followed by 1 h incubation at room temperature with 1 in 1000 dilution of goat anti-rabbit antibodies conjugated to Alexa Fluor 488 (Life Technologies). Control sera were collected from rabbits prior to immunization with rMHJ_0461. Coverslips were mounted in VECTASHIELD onto microscope slides and imaged using an Olympus BX51 Upright Epi Fluorescence microscope. Images were captured using an Olympus DP97 Digital Microscope Camera coupled with Olympus DP Controller software.