Alexa fluor 568 conjugated donkey anti rat igg

Aforementioned cryosections (4 μm) were placed on slide glasses (refer to the text of H&E staining). The sections were incubated with either 3% normal goat serum or 3% normal donkey serum and 0.5% Triton X-100 in PBS for 30 min to block non-specific binding, and then incubated with primary antibodies diluted in blocking solution overnight at 4 °C (see Supplementary Table S1 online). The sections were then incubated with secondary antibodies for 1 h (1:1000 dilution) at room temperature (22–25 °C). Alexa Fluor-488-conjugated donkey anti-goat IgG, Alexa Fluor-488-conjugated goat anti-rat IgG, Alexa Fluor-488-conjugated goat anti-chicken IgG, Alexa Fluor-568-conjugated donkey anti-rat IgG, Alexa Fluor-647-conjugated donkey anti-chicken IgG, Alexa Fluor-568-conjugated donkey anti-rabbit IgG (all 1:500 dilution; Invitrogen, USA) were used as secondary antibodies. After washes with PBS, the immunolabeled sections were mounted using PermaFluor aqueous mounting medium (PermaFluor, Thermo Scientific, USA) and were observed under a CLSM (FV1000, Olympus, Japan) with following acquisition parameters: excitation at 473 and 559 nm, × 60 water immersion lens (NA = 1.2), image size = 105 × 105 μm.

We have previously reported the following protocols of the present study^{24 (link)}. Cryosections with a thickness of 50 μm were prepared using a CM1950 cryomicrotome (Leica, Wetzlar, Germany) and immersed in PBS. The floating sections were blocked via treatment with 3% normal goat serum, 3% normal donkey serum, and 0.5% Triton X-100 in PBS for 60 min, followed by treatment with the primary antibody diluted in blocking solution overnight at 4 °C (Supplementary Table S1). This was followed by rinsing three times with PBS. The sections were then treated with a secondary antibody for 1 h (1:500 dilution) at room temperature (22 °C–25 °C). Alexa Fluor-488-conjugated donkey anti-goat IgG, Alexa Fluor-568-conjugated donkey anti-rat IgG, and Alexa Fluor-568-conjugated donkey anti-rabbit IgG (1:500 dilution; Invitrogen, Waltham, MA, USA) were used as secondary antibodies. After rinsing with PBS, the sections were mounted using PermaFluor mounting medium (Thermo Fischer Scientific, Shandon, PA, USA) and observed under a CLSM (FV1000, Olympus, Tokyo, Japan) with the following acquisition parameters: excitation at 473 and 559 nm, 60× oil immersion lens (NA = 1.2), and image size = 105 × 105 μm. The Z-stack sizes varied between 50 and 80 images. Image deconvolution and 3D reconstruction were performed using the resulting image stacks analyzed using Avizo 9.1.1 (FEI, Burlington, MA, USA).