Alexa fluor 488 conjugated anti rabbit secondary

Parasites at different stages of differentiation were centrifuged at 5,000 x g for 2 minutes, washed twice in 1x PBS, resuspended at a density of 10⁶ cells, and fixed with 4% paraformaldehyde in 1x PBS for 10 minutes. The cells (10⁶ parasites) were added to Teflon delimited field slides pretreated with poly-L-lysine and incubated in a humid chamber for 10 minutes. The cells were washed twice with 1x PBS, and this process was repeated in all the following steps. The cells were permeabilized with 0.1% Triton X-100 for 2 minutes and blocked with 1% PBS/BSA for 16 hours at 4°C, and this solution was also used to dilute antibodies. The parasites were incubated with the primary antibody for one hour at room temperature and washed three times by immersion in 1 x PBS for 5 minutes. The secondary antibody was added, and the incubation and washing steps were repeated. The cell nucleus and the kinetoplast were stained for 10 minutes with DAPI (1 g/mL) diluted in blocking solution. After this step, the slides were washed five times in 1x PBS, and 10 μL of n-propyl gallate was added. The slides were sealed with coverslips and observed with a fluorescence microscope. The serum dilutions were as follows: anti-TcNRBD1 1:300, Alexa Fluor 488 conjugated anti-rabbit secondary (Invitrogen®—Carlsbad, Califórnia, EUA) 1:400, and DAPI (4',6-diamidino-2-phenylindole dihydrochloride) 1:1000.

Pyrosomes were fixed in 4% paraformaldehyde in PBS for 20 min and then incubated in 0.5% Triton in PBS for 1 h at room temperature on a rocking nutator. Samples were then blocked in PBT-BSA (0.5% Triton X-100, 0.5% bovine serum albumin) for 30 min at room temperature. An anti-Renilla luciferase antibody (1:250, GTX125851, GeneTex) or rabbit pre-immune serum (1:250, ab37415, Abcam) was added and samples were incubated at 4 °C overnight. GTX 125851 is a polyclonal antibody with specificity for Renilla luciferase. Samples were then washed in PBS and incubated with AlexaFluor-488-conjugated anti-Rabbit secondary (1:200, Invitrogen) for 1 h at room temperature. Following secondary antibody incubation, samples were washed in PBS and Hoechst 33342 (Molecular Probes, 1:2000) was added during a 10 min wash in PBS. Samples were dissected and placed in a glass-bottomed petri dish with PBS for imaging. In addition to the pre-immune serum control, other control samples were incubated without primary or secondary antibody, and no specific signal was observed (data not shown).