Prolong gold antifade with 4 6 diamidino 2 phenylindole dapi

TA muscles from each mouse line were cryopreserved in melting isopentane for 30 s and 10 µm transverse cryosections were obtained (Leica CM3050 S). For histology, sections were stained with H&E and imaged on a Leica DM5500 B microscope equipped with a Leica HC PLAN APO 20× objective. Centrally nucleated fibers (CNFs) were counted using the Cell Counter plugin on ImageJ software (NIH) and expressed as a percentage of the total number of myofibers (%CNFs). For immunofluorescence, sections were fixed in 4% paraformaldehyde in PBS at RT for 10 min and subsequently washed three times in PBS before being permeabilized in 0.1% Triton X-100 in PBS for 10 min at RT. Sections were then blocked in 5% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS for 1 h at RT before incubating with primary antibodies overnight at 4 °C. Slides were then washed three times in PBS before incubating with Alexa Fluor secondary antibodies (1:500 each) for 1 h at RT. Sections were finally wash three times in PBS and mounted in ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (ThermoFisher Scientific). Images were acquired on a Deltavision PersonalDV deconvolution microscope equipped with an Olympus UApo 20x objective.

Non-induced and induced B. bovis cultures were collected at 0, 12, 24, and 48 h and used for IFA. Cells were washed with 1X PBS at 900xg and pellets suspended with 1X PBS containing 3% bovine serum albumin (BSA). A smear was made by taking 5 μl from each cell preparation to a glass microscope slide. The slides were air dried, fixed for 5 min in cold acetone, and blocked with 1X PBS containing 10% BSA for 30 min. Then, slides were incubated with rabbit anti-CCp5 or -FNPA sera (1:50) in 1X PBS-10% BSA for 1 h. The slides were washed 3 times with 1X PBS and incubated in 10% BSA with goat anti-rabbit IgG AlexaFluor^®555 (Thermo Fisher, Waltham, MA, USA), and then again washed 3 times with 1X PBS and mounted with a drop of Prolong™ Gold Anti-fade with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher, Waltham, MA, USA) and cover slip. The slides were analyzed using a Leica Sp8-X White Light Laser point scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).