Goat anti mouse igg h l

Proteins (60 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and electrotransferred to a nitrocellulose membrane (IPVH00010, Millipore, Burlington, MA, USA). After blocking in 5% milk at RT for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-60S ribosomal protein L18 (Rpl18) (1 : 2000, Cat. No. ab241988, Abcam, Cambridge, MA, USA), rabbit antieukaryotic translation initiation factor 3 subunit (Eif3c) (1 : 2000, Cat. No. 2068, Cell Signaling Technology (CST), Danvers, MA, USA), rabbit anti-Ras homolog family member C (Rhoc) (1 : 2000, Cat. No. 3430, CST), rabbit anti-G protein subunit gamma 13 (Gng13) (1 : 500, Cat. No. PA5-70258, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-Gng10 (1 : 500, Cat. No. PA5-97046, Thermo Fisher Scientific), and mouse anti-β-actin (1 : 1000, Cat. No. ab8226, Abcam). Next day, membranes were incubated with secondary antibodies goat anti-rabbit IgG (H + L) (1 : 5000, Cat. No. 31210, Thermo Fisher Scientific) and goat anti-mouse IgG (H + L) (1 : 5000, Cat. No. 31431, Thermo Fisher Scientific) for 1 h. Proteins were detected by enhanced chemiluminescence.

Immunogenicity of the vaccines was determined by enzyme-linked immunosorbent assay (ELISA) of sera samples collected on Day 35. HA1 and HA0 influenza B HA recombinant antigens were applied to 96-well plates and incubated overnight at 4°C (1.25 μg/mL). Plates were washed (405 TS ELISA Plate Washer, Agilent Technologies) with PBS plus 0.1% Tween 20 between all steps. After blocking (1% Omniblok, AmericanBio, Inc., and 0.1% Tween 20 in PBS), serum samples from vaccinated mice were plated at a 1:125 dilution followed by two-fold dilutions. Plates were then incubated at 37°C with a HRP conjugated secondary antibody (goat anti-mouse IgG (H+L), Thermo Fisher Scientific). Colorimetric detection occurred at room temperature and measured for absorbance at 405 nm (1-Step ABTS, Thermo Fisher Scientific). Endpoint titers levels were statistical defined per plate (31 (link)) and analysis of variance (ANOVA) tests were conducted with Tukey’s multiple comparisons test to determine group differences.

For histological examination, hearts were fixed with 4% paraformaldehyde in PBS for Sirius red staining or HE staining and cryosectioned for immunostaining. For immunostaining, fixed hearts were immersed in 30% sucrose overnight, embedded in an optimal cutting temperature compound, frozen, and cryosectioned (4.5-μM sections). Immunostaining was performed, and primary antibodies were used at dilutions of 1:200 for anti-sarcomeric alpha-actinin (Abcam, ab137346) and 1:100 for anti-GFP (Abcam, ab1218). Secondary antibodies, including goat anti-rabbit IgG (H + L) (Alexa Fluor Plus 555, Invitrogen, A32732), were used at dilutions of 1:500 for detecting alpha-actinin, and goat anti-mouse IgG (H + L) (Thermo Fisher, F2761) was used at dilutions of 1:500 for detecting eGFP. Slides were imaged with an inverted fluorescence microscope (Leica inverted microscope DMi8).

Western blot analysis was performed as previously described^{38 (link)}. Briefly, 30 or 40 μg of total protein were separated by 10% denaturing SDS–PAGE and transferred onto 0.45 μm nitrocellulose membranes overnight. The membranes were incubated with primary ß-Catenin (6B3) (1:1000, Cell Signaling, #9582), CD133 (1:250, Miltenyi, #130-092-395), p-AKT (Ser 473) (D9E) XP (1:1000, Cell Signaling, #4060), AKT (1:1000, Cell Signaling, #9272), p-MEK1/2 (Ser217/221) (1:1000, Cell Signaling, #9121), or MEK1/2 (1:1000, Cell Signaling, #9122) antibodies overnight at 4 °C. Secondary horseradish-peroxidase-coupled antibodies goat-anti-mouse IgG (H + L) (1:10,000,Thermo Fisher Scientific) or anti-rabbit IgG (H + L) (1:10,000, Thermo Fisher Scientific), and anti-biotin, HRP-linked antibody (1:5000, Cell Signaling) were then applied for 1 h at RT. Hybridization with GAPDH-HRP (6C5) (1:10,000–20,000, Abnova, #MAB5476) coupled antibody was performed for 30 min at RT as a housekeeping gene. Antibodies were visualized using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate kit (Merck Millipore). Images were generated using Gene Genome Syngene Bio Imaging and quantified using Image J (Rasband, W.S., U.S. National Institutes of Health).

A pyrimethamine (Sigma-Aldrich) stock solution was prepared at 10 mM in DMSO and was used at the final concentration of at 20 μM as a positive control for anti-Toxoplasma activity. Forty-six molecules were tested to determine their anti-Toxoplasma activity. Each molecule was prepared as a 10 mM stock solution in DMSO and tested at 10 μM. The hits were then further tested at the dilutions 0.01 to 100 μM. Hoechst-33342, trihydrochloride, trihydrate (Sigma-Aldrich) was used as a marker of nucleic acids to detect parasites and cell host nuclei. Monoclonal antibody TG17.43 anti-GRA1 (Biotem) and goat anti–mouse IgG (H+L) coupled to Alexa Fluor-488 (Thermofisher) were used to detect Toxoplasma parasites and their parasitophorous vacuole. [³H]-uracil was used to analyze parasite proliferation.