Cy3 conjugated anti rabbit immunoglobulin g igg

Rats were euthanized with Euthasol euthanasia solution and transcardially perfused with ice-cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Brains were postfixed for 4 h at 4°C and cryoprotected in 30% sucrose in 0.1 M PBS. Serial coronal sections throughout the hippocampus were cut at a 40 μm thickness using a cryostat and stored in PBS. The sections were incubated with blocking buffer (5% bovine serum albumin, 0.3% Triton X-100 and PBS) for 1 h followed by overnight incubation with mouse anti-5-HT2A receptor antibody (1:500, Cat. PC176, Millipore) at 4°C. The sections were rinsed and transferred to Cy3-conjugated anti-Rabbit immunoglobulin G (IgG; 1:500, Cat. 111-165-144, Jackson ImmunoResearch) for 2 h at room temperature. The sections were examined using a confocal laser-scanning microscope (FV-1000, Olympus, Tokyo, Japan).

Retinal sections and whole mount preparations were prepared following a previously described method (Tao et al., 2015 (link)). For immunohistochemistry, the peanut agglutinin (PNA) conjugated to a Alexa Fluor 488 (1:200, Invitrogen, USA), S-cone opsin, or M-cone opsin antibodies (1:400, Millipore, MA, USA) were incubated with retinal specimen, respectively. After thorough rinses with PBS, the retinal specimens were incubated in Cy3-conjugated anti-rabbit immunoglobulin G (IgG) (1:400, Jackson ImmunoResearch Laboratories, USA) and 4′,6-diamidino-2-phenylindole (DAPI). Cone cells within four 420x420 μm bins surrounding the ONH were quantified using AxioVision Rel. software.

Serial transverse cryosections (7-μm thick) of the midbelly region of the frozen soleus and plantaris muscles were cut at −20°C and mounted on slide glasses. The sections were air dried and stained to analyze the translocation of HSP72 into the nucleus following a standard immunohistochemical technique. Cross sections were fixed with paraformaldehyde (4%) and then were post-fixed in ice-cold methanol. After blocking by using a reagent (1% Roche Blocking Reagent, Roche Diagnostics, Penzberg, Germany), samples were incubated with primary antibodies for HSP70 (diluted 1:200; ab79852, Abcam, Cambridge, UK). Sections were also incubated with secondary antibodies for Cy3-conjugated anti-rabbit immunoglobulin G (IgG) (diluted 1:200; Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. Nuclei were then stained in a solution of 4′,6-diamidino-2-phenylindole dihydrochloride (Dapi, 1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. The images of muscle sections were incorporated into a personal computer (Keyence BZ-X viewer) by using a microscope (BZ-X700, Keyence, Osaka, Japan).