Tcs sp2 laser scanning spectrum confocal system

Mice were sacrificed 24 or 48 h pi and mammary tissues were bisected for histology and total RNA extraction. Samples for histopathological analysis were fixed overnight in neutral buffered 4% PFA, at 4 °C and embedded in paraffin, and sections were cut at a thickness of 5 µm and stained with hematoxylin and eosin (H&E) according to standard procedures. For fluorescence staining, mammary tissues were fixed in 4% PFA overnight at room temperature and incubated with 15% (w/v) sucrose in PBS for 72 h at 4 °C. The 13-µm cryosections were stained with 1 µg/mL phalloidin-iFlour 647 (Abcam) and 25 µg/mL DAPI (Sigma-Aldrich, Rehovot, Israel). M. bovis cells were visualized using polyclonal-anti M. bovis antibody (Mycoplasma collection, University of Florida, Gainesville, FL, USA) and rabbit anti-goat IgG (Sigma-Aldrich, Rehovot, Israel) applied as the secondary antibody. The mammary glands were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and imaged with an Axio Imager M1 208 upright fluorescence microscope (Zeiss, Germany). Confocal images were acquired using a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems, Wetzlar, Germany) and images were merged using Leica confocal software.

Mammary samples for histological analysis were prepared as previously described [8 (link), 14 (link)]. Briefly, paraffin sections were stained with haematoxylin and eosin (H&E) and cryosections were stained with phalloidin-TRITC (Sigma) to visualize filamentous actin (F-actin) and 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) to visualize nuclear DNA.
Gall bladder and urinary bladder whole mounts were pinned onto a silicone pinning pad (SYLGARD 184 Dow Corning), fixed with 2.5% formaldehyde and stained with phalloidin-TRITC and DAPI or WGA Alexa Flour 633 (ThermoFisher W21404) and Sytox Orange (Invitrogen) as previously described [21 (link)].
Tissue sections were imaged with a Nikon Eclipse E400 epifluorescence microscope with an Olypus DP70 camera and images were merged using Adobe Photoshop software. Confocal images were acquired using a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems) and images were merged using Leica confocal software.