Peroxidase conjugated goat anti mouse igg

MRC-5 fibroblasts were stimulated with Gal-9 or TGF-β for 48 h. Total protein from the cells was extracted by adding protein lysis buffer to the cells. Western blotting was conducted using primary antibodies of rabbit polyclonal anti-smooth muscle actin (SMA) (1:1000 dilution; Proteintech) and mouse monoclonal anti-GAPDH (1:1000 dilution; Abcam), followed by secondary antibodies including peroxidase-conjugated goat anti-mouse IgG (1:5000 dilution; Abcam) and peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution; Abcam). Enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, United States) was added to the membranes. Quantitative protein densitometry was performed with ImageJ software (NIH, Bethesda, MD, United States).

Membranes were incubated with 5%-10% fat-free milk in TBST. After blocking, membranes were incubated with the indicated primary antibodies at 4 °C overnight. Membranes were washed in TBST for 3 times and mixed with the appropriate secondary antibodies for 1 h at 37 °C. After incubation, membranes were washed in TBST for 3 times, subjected to enhanced chemiluminescence and finally observed under molecular imager ChemiDoc XRS + system. Antibodies used in this study were as follows: anti-HES1 (Abcam), anti-β-Tubulin (Abcam), anti-NOTCH1 (Cell Signaling Technologies), anti-MAML1 (Cell Signaling Technologies), anti-APH1A (Abcam), peroxidase-conjugated goat anti-mouse IgG and peroxidase-conjugated goat anti-rabbit IgG (Abcam).

Following resolution by 10% SDS-PAGE, each purified protein sample was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using eBlot™ L1 (GenScript, Nanjing, China). The membranes were separately incubated at 37 °C for 2 h with mouse anti-His tag monoclonal antibody (CWBIO, Beijing, China), chicken anti-E. tenella serum, and normal chicken serum. After washing with TBST buffer, the membranes were separately incubated at 37 °C for 1 h with the corresponding secondary antibodies: peroxidase-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-chicken IgY (Abcam, Cambridge, UK). The binding of secondary antibodies was revealed by the enhanced HRP-DAB chromogenic substrate kit (TIANGEN, Beijing, China).
Protein extracts of merozoites were obtained by using RIPA lysis buffer (Beyotime, Nantong, China). Western blot analysis was carried out using mouse anti-rEtROP17 serum as the primary antibody. Goat anti-mouse IgG conjugated with horseradish peroxidase (Abcam, Cambridge, UK) was used as a secondary antibody. The bound antibody was detected using protein enhanced chemiluminescent (ECL) reagent (Thermo Scientific, Waltham, MA, USA). The slide stained with the serum before immunization as the primary antibody was used as the control.