Hrp conjugated goat anti rabbit igg

The PMVECs were lysed with cell lysates and then separated by SDS-polyacrylamide gel electrophoresis (Solarbio, Beijing, China) and transferred onto polyvinylidenedifluoride membranes (Millipore, USA). The samples were incubated with anti-rabbit Acod1 antibody (1:1000; Abcam, Shanghai, China) or anti-rabbit Nrf2 antibody (1:1000; ABclonal, Shanghai, China) at 4℃ overnight. Anti-rabbit Histone H3 (1:5000; Gene Tex, USA) or anti-mouse GAPDH (1:10000; Proteintech, Wuhan, China) were used as the controls. Subsequently, these cells were subjected to incubation with HRP-conjugated goat anti-rabbit IgG (1:3000; Solarbio, Beijing, China) or HRP-conjugated goat anti-mouse IgG (1:3000; Solarbio, Beijing, China) at 37℃ for one hour. The proteins were visualized by enhanced chemiluminescence (Solarbio, Beijing, China).

Thin smears of T. brucei were fixed in cold methanol for 10 min before blocking in 5% skim milk. The mouse anti-TryR antibody was used as the primary antibody at a dilution of 1:50. Alexa Fluor 594-conjugated goat anti-mouse IgG (SE134; Solarbio, Beijing, China) was used as the secondary antibody at a dilution of 1:600. The nuclei of the parasites were stained with DAPI. T. brucei parasites that were not exposed to GQDs were used as controls. Serum from an unimmunized mouse and PBS buffer without antibodies were used as the negative and blank controls, respectively. High-resolution images were captured using a confocal laser scanning microscope (SP8; Leica, Wetzlar, Germany).
The total lysate of T. brucei that received GQDs treatment was mixed with the SDS-PAGE loading buffer, and the proteins were separated using 12% SDS-PAGE. Subsequently, the proteins were transferred onto PVDF membranes. Before co-incubation with mouse anti-TryR IgG (1:1,000), the membranes were blocked with 5% skim milk at 37°C for 1 h. HRP-conjugated goat anti-rabbit IgG (SE134; Solarbio, Beijing, China) was used as the secondary antibody. Signals obtained with rabbit anti-GAPDH IgG were used for normalization.

At 0, 7, 14, 21, 28 and 35 days after the first immunization, blood was sampled from auricular veins of rabbits for antibody level analysis. In the immune sera, OD values at the same dilution were measured by ELISA, which reflected the antibody level [18 (link)]. In all ELISA tests, the 96-well microplates were coated with 1 µg of rPmy in 1.0 M carbonate buffer (100 μl/well), pH 7.4, at 4 °C overnight and then blocked with 10% bovine serum albumin in PBS with Tween-20 (PBST) (100 μl/well) at 37 °C for 1 h. After washing three times with PBST, microplates were incubated with rabbit serum (100 μl/well) at 37 °C for 1 h. After washing three times with PBST again, microplates were incubated with HRP-conjugated goat anti-rabbit IgG (Solarbio, Beijing, China), diluted 1:10,000 in PBST, at 37 °C for 1 h (100 μl/well). TMB colored liquid was added to the microplates and incubated at 37 °C for 15 min in darkness. The reaction was stopped by adding 1.0 M H₂SO₄ (50 μl/well). Subsequently, the absorbance at a wavelength of 450 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).