Carl lsm 710 confocal microscope

The expression changes of representative IL-17 (CgIL17-1) (57 (link)) at the protein level in RGD⁺ hemocytes after V. splendidus challenge were analyzed by immunofluorescence assay. In brief, resting RGD⁺ hemocytes and activated RGD⁺ hemocytes were plated onto glass-bottom culture dishes for 1 h of cell adhesion. Cells were washed with PBS and fixed with 4% PFA at room temperature for 15 min before they were permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with 3% BSA in PBS-Tween for 1 h and incubated with the mouse antiserum against CgIL17-1 (46 (link), 58 (link)) previously prepared in our lab (1:100-diluted in PBS-Tween) at room temperature for 1 h. Then, hemocytes were extensively washed with PBS-Tween and incubated with Alexa Fluor 594-labeled goat anti-mouse IgG (1:500-diluted in PBS-Tween) for 1 h, followed by incubation in DAPI (US Everbright, Inc., Greenfield, WI, USA) for 10 min. The hemocytes were washed, and fluorescence images were captured using a Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Germany). The red fluorescence intensity of Alexa Fluor 594 was analyzed by Zeiss software to reflect the changes in CgIL17-1 protein expression. The unimmunized mouse serum and commercial Alexa Fluor 594-labeled goat anti-mouse IgG were used as the controls for the primary and secondary antibodies, respectively.

Embryo fixation was performed according to standard methods. Embryos were fixed by a solution containing heptane (Sigma, St Louis, MO) and methanol^{57 (link),58 (link)}. Wing discs were fixed in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 35 mM phosphate buffer, pH7.4) for 15–30 min at room temperature.
Antibodies used for immunohistochemistry were as follows: mouse anti-Cut (1:200, 2B10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), guinea pig anti-Centrosomin (1:1000, from Jordan Raff), Rat anti-α-Tubulin (1:200, MAB1864, Millipore, Burlington, Massachusetts), rabbit anti-PH3 (1:200, 06–570, Millipore), Rabbit anti-Vtd (1:500, this study), rabbit anti-Myc (1:100, ab9106, Abcam, Cambridge, UK). Secondary antibodies conjugated with Rhodamine Red™-X (RRX), Alexa Fluor® 647 or fluorescein isothiocyanate (1:200, 715-095-151, 715-295-151, 715-605-151, 711-095-152, 711-295-152, 711-605-152, 706-095-148, 706-295-148, 706-605-148, 712-095-153, 712-295-153, 712-605-153) were from Jackson ImmunoResearch Inc. Vectashield with 4′, 6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The subcellular localization of ApLAO was determined by the fluorescent molecule FITC. FITC (3 mg) was added to the purified ApLAO in 0.1 M Na carbonate buffer, pH 11, and incubated 3 h at room temperature with constant stirring. Unbound FITC was removed by dialysis against PBS supplemented with 5 g/l activated carbon, followed by dialysis against PBS in the dark. FITC-labeled ApLAO was concentrated and filter-sterilized. Jurkat cells were seeded into a 24-well culture plate in duplicate (2×10⁵/well) and treated with the ApLAO-FITC (5 μg/ml) for 1, 10 and 60 min. The cells were then cytospun on the slides for 6 min at 1300 r.p.m. and fixed in 10% (w/v) formalin in PBS (pH 7.4) for 15 min at room temperature. After washing with PBS, Prolong Antifade Kit (Molecular Probes) was mounted on dried slides and allowed to dry overnight at 4 °C. A Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany) with ZEN 2011 image software (Carl Zeiss) was used for fluorescence microscopy.