Living colors anti rcfp polyclonal pan antibody

In adult Nile tilapia, the only non-surgical access to the testis is via the common spermatic duct that opens in the urogenital papilla through the urogenital pore. Ten 6-month-old male fish were anesthetised and received 300 μL of DsRed2 lentiviral particle solution (6.5 × 10⁵ TU) through the common spermatic duct using a glass micropipette (outside diameter 70 μm) under a stereomicroscope (OLYMPUS SZX-ILLB2-100)26 (link). They were maintained for 1 week in tank water at 30 °C to stimulate spermatogenesis27 . The gonads were removed in duplicate 24 h, 3.5 d and 7 d after injection, and immersed in Tissue-Tek O.C.T. Embedding Compound (SAKURA FINETEK). Cryomicrotome sections were washed in PBS, stained using DAPI (SIGMA), and then analysed by fluorescence microscopy in order to verify the red fluorescent protein expression in the fish testis. Twenty-four hours and 7 d after lentiviral injection, sperm were also collected by abdominal massage and submitted to flow cytometry. mRNA obtained from the fish testis and isolated sperm was analysed by RT-PCR as described above. Immunohistochemistry was also performed in transduced Nile tilapia testis using Living Colors Anti-RCFP Polyclonal Pan Antibody (CLONTECH) and anti-DDX4/Vasa (ABCAM, 1:400). Secondary Alexa Fluor488 donkey anti-Rabbit IgG (INVITROGEN, 1:500) was used for fluorescent detection.

H1299 cells that were transfected with YFP-RhoA or the YFP-vector (pZsYellow1-C1) were lysed in RIPA buffer, boiled in Laemmli sample buffer and subjected to SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and immunoblotted using an antibody against pZsYellow (Living Colors Anti-RCFP polyclonal pan antibody) from Clontech (Mountain View, CA). Bound antibodies were visualized using horseradish peroxidase-linked anti-rabbit IgG (Santa-Cruz Biotechnology, sc-2004) and ECL reagents (Bio-Rad) per manufacturer's recommendation.

Fluorescent proteins were detected from whole cell lysates. S. aureus was cultured overnight as described in the growth conditions. The pre-cultured cells were adjusted to an optical density at 660 nm of 0.02 with TSB with chloramphenicol, and 3 ml of the culture were transferred into test tube (25 mm × 150 mm) and then incubated at 37 °C with shaking at 140 rpm for 16 h. The bacterial cells from 1 ml cultures were harvested by centrifugation, and then the whole cell lysates were prepared as follows: cells were re-suspended in 200 μl CS buffer (100 mM Tris-HCI, 150 mM NaCl, 100 mM EDTA, pH7.5) containing 1 μg of lysostaphin (Wako Pure Chemical Industries, Co., Ltd, Japan) and incubated at 37 °C for 30 min. Ten microliters of cell lysates were separated on SDS–PAGE and stained with Coomassie Brilliant Blue (CBB), and the fluorescent proteins were detected using Western blot with the following antibodies: Anti-GFP-HRP-Direct T (MBL Co., Ltd.), Living colors® Anti-RCFP Polyclonal Pan Antibody (Clontech Laboratories) and DsRed Polyclonal Antibody (Clontech Laboratories) as the primary antibody, and HRP-conjugated goat antibodies against rabbit IgG (MP Biomedicals, LLC-Cappel Products) as the secondary antibody. Immuno-detection of protein was performed on Pierce® Western Blot Substrate (Thermo Fisher Scientific Inc.) with X-ray film.