Goat anti rabbit igg alexa fluor plus 488

Protein expression and cell distribution of FL-(P)RR in cultured podocytes were assessed through immunofluorescence (IF) analysis. Cells were fixed in 4% paraformaldehyde. Subsequently, a subset of cells for each condition underwent pretreatment with triton X-100 to enhance antibody penetration, while another subset remained untreated to preserve cell membrane integrity. Following this, cells were blocked with 10% normal goat serum for 30 min at room temperature (RT). The cells were then incubated overnight at 4 °C with the #HPA003156 antibody (Sigma, 1:75). This was followed by a 1 h incubation at RT with a fluorochrome-conjugated secondary antibody (Goat anti-rabbit IgG AlexaFluor Plus 488, Thermo Fisher Scientific, #A32731, 1:500). Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific, #3570, 1:2000). IF images were captured using a 40x/0.55 Ph1 objective on a Zeiss Axiovert 200 M fluorescence microscope equipped with an Axiocam 503 color camera, controlled by ZEN 2.0 (blue edition) software (Zeiss, Milan, Italy).

BrdU immunohistochemistry were performed as previously described (Ma et al., 2008 (link)) with the following modifications for double immunostainings: fish were rinsed, permeabilized and processed for blocking then incubated overnight at 4°C in blocking solution containing two primary antibodies: Anti-DsRed rabbit (Living Colors^® DsRed Polyclonal Antibody, Catalog #: 632,496) at 1:500, Anti-BrdU from mouse IgG1 (Roche, Catalog #: 11170376001) at 1:100. After extensive washes at room temperature, the larvae were incubated in blocking solution containing a mix of secondary antibodies: Goat anti-Rabbit IgG Alexa Fluor Plus 488 (Thermofisher, Catalog #: A32731) and Goat anti-Mouse IgG, Alexa Fluor Plus 594 (Thermofisher, Catalog #: A32742) diluted at 1:500.
To perform whole mount ISH, larvae were fixed at desired stages in 4% PFA in PBS overnight at 4°C then processed through standard protocol (Nguyen-Chi et al., 2015 (link)).

For detecting IZUMO1 in sperm cells, the frozen rat testes were disaggregated in PBS using BioMasher II (Nippi, Tokyo, Japan), and the germ cells were smeared on glass slides. The cells were air-dried on microscope slides, washed with PBS, and permeabilized with methanol. After treatment with image-iT FX Signal enhancer (Thermo Fisher Scientific) for 30 min at room temperature, the cells were incubated with the rabbit anti-human IZUMO1 polyclonal antibody (1:100) diluted by Can Get Signal A (Toyobo) at 4°C for overnight. After incubation with goat anti-rabbit IgG Alexa Fluor Plus 488 (1:200) and Lectin PNA Alexa Fluor 568 conjugate (Thermo Fisher Scientific) (1:200) for 1 h at room temperature, the spermatozoa were stained with Hoechst 33342 to visualize the nuclei (Dojindo, Kumamoto, Japan) and were mounted in Prolong Diamond (Thermo Fisher Scientific). For sperm morphological analysis, the cauda epididymal spermatozoa of male rats were incubated in the Human Tubal Fluid (HTF) medium (Aoto et al., 2011 (link)) for 10 min and observed under a BX50F phase-contrast microscope (Olympus, Tokyo, Japan).