Alexa fluor 594 goat anti rabbit igg

On day 8 after the cystometry study, animals were euthanized with isoflurane, and the bladders were harvested. Each bladder was halved for histological, immunofluorescent, and Western blot analyses. For histologic analysis, bladder tissues were fixed in 4% paraformaldehyde for 24–48 h, followed by embedding in paraffin. Four-micrometer sections were stained with hematoxylin and eosin.
Immunofluorescence staining of tissue sections involved blocking 4-μm sections with goat serum, incubating them with primary antibodies overnight at 4°C, and then incubating them with Alexa Fluor 488 Goat Anti-Rabbit IgG (1:200, Yeasen, China) or Alexa Fluor 594 Goat Anti-Rabbit IgG (1:200, Yeasen, China) for 1 h at 37°C. After DAPI staining, fluorescent images were captured using a fluorescence microscope.

To verify PrRP cellular expressing distribution and functional region in grass carp, the hypothalamus of grass carp was isolated, obtained, and fixed in 4% paraformaldehyde. After fixation, the hypothalamus was dehydrated by gradient ethanol, then hyalinized in xylene and embedded with paraffin wax. The hypothalamus was sectioned under the condition of maximum longitudinal section and a thickness of 4 μm by a slicer (LEiCA, Shanghai, China). In the immunofluorescence experiment, after dragging wax and rehydration, the tissue section was soaked in citric acid antigenic repair buffer and microwaved from high to medium heat to accomplish antigen retrieval. To block nonspecific sites, the sections were incubated with 5% normal goat serum (Yeasen) for 1h at ambient temperatures. The sections were incubated with PrRP1 antibody (1:800) overnight at 4°C. Then after washing sections five times with 1×PBS the second day, added Alexa Fluor 594 Goat Anti-Rabbit IgG (1:200) (Yeasen). Finally, the section was subsequently counterstained with DAPI and sealed with an anti-fluorescence quenching mounting medium. The stained sections were imaged with a Leica SP8 confocal microscope.