Goat anti mouse alexa 594

The various tissues were fixed in 10% neutral buffered formalin and afterwards embedded in paraffin. For immunohistochemistry, tissue sections (4 μm thickness) were deparaffinized, rehydrated and subjected to heat-induced antigen retrieval in Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH 9). The sections were permeabilized with 0.5% Tween 20 in PBS for 15 min at room temperature and blocked with 5% BSA, 5% goat serum in PBST (0.05% Tween) for 90 minutes at room temperature. Slides were then incubated with the primary antibodies overnight at 4°C. After 3 washes with PBST for 5 min, the sections were incubated in the dark with the secondary antibodies. For counterstain DAPI (1 μg/ml) was used and Mowiol 4–88 (Polysciences) was used for mounting. Primary antibodies: mouse anti-VCP (1:250, Abcam, ab11433), rabbit polyclonal anti-K315me3 (1:500, New England Peptides, custom antibody against synthetic peptide H2N-AIAPKRE(3me)KTHGEVERR-OH, double affinity purified), anti-VCPKMT (1:500, serum of rabbits immunized with recombinant human VCPKMT [6 (link)]). Secondary antibodies: goat anti-rabbit-Alexa 488 (1:500, Life Technologies), goat anti-mouse-Alexa 594 (1:500, Life Technologies). Images of fluorescently stained sections were acquired using AxioCam MRRev3 camera on an Axio Observer. Z1 microscope (Carl Zeiss).

Smears of parasite cultures on glass slides were fixed in ice cold 10% methanol/90% acetone for 10 min. Slides were rehydrated for 10 min in PBS/3% BSA and then incubated for 2 hours with primary antibodies in PBS/3% BSA. After three washes with PBS, the slides were incubated with secondary antibodies and Hoechst 33342 for 2 hours. After washing with PBS, the slides were mounted with Prolong Antifade mounting medium (Life Technologies) and left to dry overnight. Imaging was conducted on an Axio Observer7 fluorescence microscope (Zeiss) using a ×100 Oil Objective and images were processed using the ZEN 2.3 software suite. Antibodies used were mouse anti-Ty1, 1:500 (Sigma Aldrich); rat anti-HA 3F10, 1:500; goat anti-mouse Alexa 594, 1:1000 (Life Technologies); goat anti-rat Alexa 488, 1:1000 (Life Technologies); Hoechst 33342 200 nM.

Cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in PBS. Primary antibodies and dilutions were used as follows: mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank) at 1:5; rat monoclonal anti-MyoD (5F11) (Millipore) at 1:100; mouse monoclonal anti-myogenin (F5D) (Developmental Studies Hybridoma Bank) at 1:5; chicken anti-Syndecan-4 [23 (link)] at 1:500; rabbit monoclonal anti-active caspase-3 (C92-605) (BD Pharmingen) at 1:100. The following secondary antibodies were used at 1:500: goat anti-mouse Alexa 594, goat anti-rabbit Alexa 594, goat anti-mouse Alexa 488, donkey anti-rat 488 (Life technologies) and donkey anti-chicken-AMCA (Jackson). Cell nuclei were stained with Hoechst 33342 (Life technologies) and Vectashield (Vector Laboratories) was used as mounting media. Images were acquired using an IX71 microscope (Olympus) equipped with a QICam FAST QImaging camera and MoticBA410 microscope with a Moticam Pro 252B camera. Images were processed using Illustrator (Adobe).

Embryos were fixed in methanol and frozen in liquid N2, as previously described (Hamill et al., 2002 (link)), then stained with 1:2000 anti-SPD-5 (non-pS530; mouse; BX23 clone) and 1:5000 anti-SPD-5 (total; rabbit; 758 clone which recognizes a.a. 1053-1198). 1:400 goat anti-rabbit-alexa488 and 1:400 goat anti-mouse-alexa594 (Life Technologies) were used as secondaries.

Rat anti-HA (100 µg/ml, clone 3F10, Roche), mouse anti-HA (1 mg/ml, clone HA.11, 16B12 Covance), rabbit anti-IFITM1-NTD (100 µg/ml, Sigma), rabbit anti-IFITM3-NTD (250 µg/ml, Abgent), rabbit anti-VDAC (1 mg/ml, Abcam), rabbit anti-calreticulin (Thermo Scientific), mouse anti-tubulin (clone DM1A, ascites fluid, Sigma), goat anti-rat Alexa-488, goat anti-rabbit Alexa-488, goat anti-mouse Alexa-594 and goat anti-rabbit Alexa-647 (all 2 mg/ml, Life Technologies), goat anti-rabbit IRDye 680 and goat anti-mouse IRDye 800 (1 mg/ml, Li-COR), and wheat germ agglutinin (WGA) conjugated to Alexa-647 fluorophore (1 mg/ml, Invitrogen), were used at the dilutions given below.