Goat anti mouse igg alexafluor594

Primary antibodies used for immunofluorescence microscopy and Western Blotting were obtained from Cell Signaling Technology and included: Beta-actin (#3700 and #4970), phospho-p70s6k T389 (#9234), NF kappa B p65 (#8242S), beta-tubulin (#2128S), Caspase-3 (#14220), Caspase-1 (#3866), Phospho-MLKL (#91689). Secondary antibodies used to detect the primary antibodies for Western Blotting were obtained from Thermo Fisher Scientific, and included goat anti-rabbit and goat anti-mouse IgG-HRP (#31460 and #31430). Secondary antibodies used for immunofluorescence microscopy were obtained from Molecular Probes (Life Technologies), and included goat anti-rabbit IgG AlexaFluor488 and goat anti-mouse IgG AlexaFluor594.

We used anti-GFP antibodies to determine the accessibility of GFP-tagged lectins in mature cyst walls. Without prior fixation, mature cysts expressing GFP-fusions under their own promoter were blocked with 1% BSA, incubated with 1:400 mouse anti-GFP IgG (Roche) for one hr at RT, washed, and then incubated with 1:800 goat anti-mouse IgG-Alexa Fluor 594 (Molecular Probes, Invitrogen). Preparations were washed, labeled with WGA and CFW, fixed in paraformaldehyde, mounted on glass slides, and observed with widefield microscopy, as described above. To determine the accessibility of glycopolymers in mature cyst walls, we used MBP-fusions to Luke(2), Leo, and Jonah(1) lectins. Mature cysts were fixed, blocked, and incubated with 15 μg of each MBP-cyst wall protein fusion conjugated to Alex Fluor 594 for 2 hr at 4°C. Preparations were labeled with WGA conjugated to Alexa Fluor 488 and CFW, as described above, and visualized with widefield microscopy and SIM. To count the number of ostioles per cyst wall, we rotated three-dimensional SIM reconstructions of mature cysts expressing Luke(2)-GFP or Leo-GFP or non-transfectants labeled with WGA, MBP-Luke(2), or MBP-Leo, all of which clearly outlined conical ostioles.

Whole cell lysates were subjected to western blot as previously described [33 (link), 34 (link)]. RAD51 immunoblot analysis was performed with the rabbit anti‐RAD51 (Merck, Darmstadt, Germany, Cat#PC130). Beta‐actin was immunoblotted by mouse anti‐beta‐actin (Sigma Aldrich, Taufkirchen, Germany, Cat#A1978) and used as a loading control. Goat‐anti‐mouse IgG‐Alexa Fluor 594 (Molecular Probes, Sigma Aldrich, Taufkirchen, Germany, Cat#A11005) and goat‐anti‐rabbit IgG‐AlexaFluor 488 (Molecular Probes, Cat#A11008) secondary antibodies were used. Membranes were developed and analyzed using LiCor Biosciences (Lincoln, NE, USA) at room temperature.