Tcs sp5 mp confocal microscope

Fat bodies of infected locusts were fixed in PBS with 4% formaldehyde, washed in PBS containing 50 mM glycine, incubated in 30% sucrose for cryoprotection, and frozen in liquid nitrogen. Frozen sections (10 μm thickness) were prepared with the Microm HM 520 cryotome and placed on microscope slides. The sections were blocked in TTBS with 1% bovine serum albumin and incubated overnight at 4°C with Abs diluted 1∶50 in blocking solution. For co-localization of parasite proteins, Abs against A. locustae Hsp70 were raised in mice. Anti-α/β-hydrolase and anti-hexokinase Abs were raised in rabbits. After washing with TTBS, slides were incubated for 2 hours at room temperature with Alexa Fluor 488 Anti-Mouse IgG and Alexa Fluor 546 Anti-Rabbit IgG Abs (Life Technologies), diluted 1∶50 in TTBS, and washed with the same solution. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The preparations in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) were observed with the help of a Carl Zeiss AxioImager M1 fluorescent microscope and a Leica TCS SP5 MP confocal microscope.

Cells were seeded on glass coverslips, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were permeabilized with Triton X-100 at 0.1% and blocked with 10% goat serum in PBS. After a first incubation with rabbit polyclonal anti-5-HT₄ receptor primary antibody (1:1000) at 4 °C overnight, cells were extensively washed and then incubated with Alexa 488 fluorophore-conjugated secondary antibodies (1:1000) for 4 h. After another wash, coverslips were mounted, and images were obtained with a Leica TCS SP5 MP confocal microscope.