After hybridization, the microarray was washed 3 times in wash buffer and then labeled for 10 min at 50°C with a solution containing streptavidin-allophycocyanin (1mg/ml final concentration), 6× SSPE, 1× Denhardt's solution, and 0.01% Tween-20.
Cells were adjusted to 100,000 per sample in 100 µl of FACS buffer and then labeled with monoclonal antibodies conjugated with a fluorescent dye [SSEA1 and SSEA3: Alexa488 purchased from eBioscience (San Diego, CA); TRA-1-60 and TRA-1-81: purchasedsed from BioLegend (San Diego, CA)].
Some of the cells stimulated with P31-43 or medium alone were treated with acid buffer (2 mM glycine and 150 mM NaCl) for 10 min at 4°C and then labelled with PE-conjugated anti-IL-15 mAb [28].
20 μg of NF-κB sense and antisense strand (Eurofins MWG Operon) were annealed and then labelled with 12 μl of gamma-P-ATP (Perkin Elmer) in kinase buffer.
For marker analysis one million testicular cells were firstly stained with Ho42 in 1 mL incubation buffer for 45 min. at 32°C, and then labeled with 1 µg of antibody (PE- anti-DR5 (MD5-1, eBioscience), or FITC-anti-human-CD49f, APC-anti-Kit and Pc5-anti-CD45 (BD Pharmingen)) for 15 min at 4°C.
In general, 1 2 × 10 cells were washed twice with PBS and then labeled with Annexin V- FITC and PI in binding buffer (JiaMei, Beijing, China), according to instructions provided by the manufacturer.
Sections were labeled overnight at 4°C with primary antibodies diluted in the blocking buffer containing 0.1% Triton X-100, washed in PBS containing 0.1% Triton X-100, and then labeled for 1 h at 37°C with a fluorescein-conjugated secondary antibody mixture in the same buffer containing Alexa-594-phalloidine (Invitrogen) F-actinctin staining.
The membranes were hybridized at 60°C for 30 min in hybridization buffer and then hybridized with labeled probes at 60°C overnight.
The membrane was pre-hybridized for 1 h with hybridization buffer (Sigma), and then the labeled probe was added and allowed to hybridize for 16 h at 37 °C.
The membranes were hybridized at 60°C for 30 min in hybridization buffer and then hybridized with a labeled probe at 60°C overnight.
