Alexa fluor 488 labeled goat anti rabbit igg

Atropine, N-nitro-L-arginine (L-NNA), magnesium sulfate and PGE₂ were
purchased from Wako Pure Chemical (Osaka, Japan). 5-HT was purchased from Tokyo chemical
industry (Tokyo, Japan). An Alexa Fluor 488-labeled goat anti-rabbit IgG and an Alexa
Fluor 568-labeled goat anti-mouse IgG were purchased from Molecular Probes Inc. (Eugene,
OR, U.S.A.). Rabbit polyclonal antibodies against NCX1 and NCX2 were produced as described
previously [9 (link)]. A mouse polyclonal antibody against
PGP9.5 was purchased from UltraClone Limited (Wellow, U.K.).

Oocytes were washed twice in DPBS-PVA and then fixed in DPBS-PVA containing 4% paraformaldehyde and 0.2% Triton X-100 for 40
min at room temperature. Thereafter, fixed samples were washed twice in DPBS-PVA for 15 min and stored overnight in 1% BSA in
DPBS-PVA (BSA-DPBS-PVA) at 4 C prior to staining. The next day, oocytes were blocked with 10% goat serum (Dako A/S, Glostrup,
Denmark) in DPBS-PVA-BSA for 45 min and then incubated in DPBS-PVA-BSA containing rabbit polyclonal anti-phospho-p38 antibody
(1:100, #9211, Santa Cruz Biotechnology) at 4 C overnight. After three washes in PBS-PVA-BSA, oocytes were incubated in
DPBS-PVA-BSA containing Alexa Fluor 488-labeled goat anti-rabbit IgG (1:300; Molecular Probes Inc., Eugene, OR, USA) for 40
min at room temperature, and then the chromosomes were stained with Hoechst 33342 (10 μg/ml, Sigma-Aldrich). Negative control
images were obtained by omitting the first antibody during staining. Following a complete washing, oocytes were mounted on
slides with mounting medium (50% DPBS, 50% Glycerol, 25 mg/ml NaN₃) and observed under an Olympus epifluorescence
microscope (AX-70). The intensity of p-p38 expression in oocytes was analyzed with the ImageJ software [17 ].

For ELISA, the tissue was rinsed with and homogenized in 1× phosphate-buffered saline (PBS). After two freeze-thaw cycles were performed, the homogenate was centrifuged at 2℃ to 8℃ for 5 minutes at 5,000×g. The levels of TRPV1 and TRPV4 in the specimen were measured according to the manufacturer’s instructions using a human TRPV1 ELISA kit and a human TRPV4 ELISA kit (Cusabio Biotech Co., Houston, TX, USA). The optical density of each well was measured within 5 minutes with a microplate reader (VersaMax; Molecular Devices Co., San Jose, CA, USA) set to 450 nm. For immunofluorescence staining, bladder tissues were fixed in 4% paraformaldehyde and rinsed with PBS. The slides were exposed to blocking solution at room temperature for 2 hours to inhibit nonspecific signal. The tissue sections were incubated with antibodies to TRPV1 (1:500; Abcam Ltd., Cambridge, UK) and TRPV4 (1:600; Novus Biologicals, Centennial, CO, USA) at 4℃ overnight. After washing three times with PBS, the slides were incubated with secondary antibody, Alexa Fluor 488-labeled goat anti-rabbit IgG (1:600; Molecular Probes, Eugene, OR, USA), at room temperature for 2 hours. Slides were stained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA), and a BX 50 microscope (Olympus, Tokyo, Japan) was used for fluorescence visualization.