Alexa fluor 488 affinipure donkey anti rat igg

The detailed information on the compounds, antibodies and reagents is listed in Supplementary Table 1. The secondary antibodies included the following: Cy3 AffiniPure Donkey Anti-Goat IgG (H+L) (705-165-147), Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (711-545-152), Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L) (715-225-151), Cy5 AffiniPure Donkey Anti-Rabbit IgG (H+L) (711-175-152), Alexa Fluor 488 AffiniPure Donkey Anti-Rat IgG (H+L) (712-545-153), Cy3 AffiniPure Donkey Anti-Rat IgG (H+L) (712-165-153), peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003) and AffiniPure Donkey Anti-Rat IgG (H+L) (712-005-153) were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

After 8 or 12 h of culture, MI- or MII-stage oocytes were isolated for the immunofluorescence assay, to assess chromosome alignment or to detect the anti-CENP-A antibody signal in oocyte from serum-ACA-positive mice.
The procedures for the immunofluorescence assay were as follows: oocytes were fixed in 4% polyoxymethylene and permeated with 0.5% Triton X-100 (Sigma, USA), followed by sealing in 5% normal donkey serum (Jackson Immunoresearch, USA). For staining of Spindle microtubules, oocytes were incubated overnight at 4 °C with rat anti-tubulin antibody (Abcam, United Kingdom, 1:800 dilution). After three washes in washing buffer, oocytes were incubated with Alexa Fluor® 488 AffiniPure Donkey anti-Rat IgG (Jackson Immunoresearch, USA, 1:500 dilution) for 1 h at room temperature, rinsed, incubated with 1 μg/mL DAPI (Cell Signaling Technology, USA) for 15 min, rinsed again, and fixed in a dish for subsequent microscopic observation. For intra-oocyte IgG staining, following incubation with Alexa Fluor® 647 AffiniPure Donkey anti-Mouse IgG (Jackson Immunoresearch, USA, 1:500 dilution) for 2 h at room temperature, oocytes were washed 3 times in washing buffer, and co-stained with 1 μg/mL DAPI for 15 min. These oocytes were mounted on glass slides and examined with a confocal laser-scanning microscope (LSM780; Zeiss GmbH, Germany).