Goat alexa fluo 488 anti rabbit igg

Cells were fixed with 3.7% formaldehyde for 15 min at RT, permeabilized with 0.05% Triton X-100 in PBS, and blocked with 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies (rabbit α-PTTG1, Abcam; mouse α-ZEB1, Santa Cruz) and then incubated with goat Alexa Fluo-488 anti-rabbit IgG and/or goat Alexa Fluo-594 anti-mouse IgG (Molecular Probes, Eugene, OR, USA).

Cells are fixed with 3.7% formaldehyde for 15 min at room temperature (RT), permeabilized with 0.05 Triton X-100 in PBS, and blocked with 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies (rabbit α-PTTG1, Abcam, Cambridge, UK; mouse α-PBF, Novus Biologicals, Centennial, CO, USA) and then incubated with goat Alexa Fluo-488 anti-rabbit IgG and/or goat Alexa Fluo-594 anti-mouse IgG (Molecular Probes, Thermo Fisher, Waltham, MA, USA). DNA was stained with Prolong Gold DAPI (Molecular Probes).

Immunofluorescence and confocal microscopy were used to detect the expression and localization of OR51E2 in human spermatozoa and HeLa cells. In HeLa cells (kindly provided by Dr. Fabiola Moretti), we overexpressed FlagRho-OR51E2 (kindly provided by Dr. Jennifer Pluznick) to verify the specificity of the α-OR51E2 antibody. Human sperm cells, purified as described above, were treated with PA 50 mM or Ringer’s solution (CTR) for 10 min at room temperature. Then, the sperm cells were washed and smeared on polylysine slides, and then allowed to air-dry. Then, HeLa and sperm cells were fixed in 3.7% formaldehyde for 15 min at room temperature (RT) and subsequently treated with 5% BSA to block nonspecific binding. Then, both cellular models were treated with polyclonal anti-OR51E2 (1:200) overnight (Aviva System Biology, San Diego, CA, USA) antibody, washed three times by PBS, and incubated for 45 min with goat Alexa Fluo-488 anti-rabbit IgG (A11034 Molecular Probes, Thermo Fisher, Waltham, MA, USA). For the HeLa cells, DNA was stained with Prolong Gold DAPI (P36935 Molecular Probes), while in sperm cells, acrosomal compartment was counterstained with lectin PNA (L32459 Molecular Probes) following the manufacturer’s instructions.