sperm immunostaining

 

Developmental Biology

Volume 306, Issue 2, 15 June 2007, Pages 797–808

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Immunofluorescence microscopy

The sperm were suspended in 10% DB and then air-dried on the slide glass. The sperm were fixed with 3.5% formaldehyde in PBS (0.9% NaCl, 10 mM Na₂HPO₄ and NaH₂PO₄, pH 7.5) at room temperature for 30 min. After washing with PBS, the sperm were post-fixed with 100% methanol at ? 30 °C for 30 min. After washing with PBS for 5 min 3 times, the samples were blocked by PBS containing 1% bovine serum albumin (BSA) in a moist chamber. The samples were treated with the anti-porcine citrate synthase rabbit polyclonal antibody against the whole molecule (1:100 dilution, NE040/7S, Nordic Immunological Laboratories B.V.) in the moist chamber at 4 °C overnight. After washing with TTBS for 5 min 3 times, the samples were treated with anti-rabbit IgG goat antibody conjugated with Alexa 546 (1:400 dilution, A-11010, Molecular Probes) for 30 min at room temperature. After washing with TTBS for 5 min 5 times, some samples were treated with anti-α-tubulin monoclonal antibody conjugated with FITC (1:200 dilution, F2168, SIGMA) at 4 °C overnight. The samples were mounted with slowfade antifade kit (S-2828, Molecular Probes). In some experiments, the sperm were incubated with 1 μM MitoTracker Green FM (M-7514, Molecular Probes) at room temperature for 1 h and then fixed and immunostained as described above. Sperm were observed under the laser-scanning microscope

 

Developmental Biology

Volume 274, Issue 2, 15 October 2004, Pages 370–383

Mammalian phospholipase Cζ induces oocyte activation from the sperm perinuclear matrix

Immunofluorescence microscopy

B6D2F₁ cauda epididymidal spermatozoa were fixed with 4% (w/v) paraformaldehyde in phosphate buffer for 15 min at room temperature (RT) and subjected to indirect immunostaining. Staining MN13 epitopes was as previously described (Manandhar and Toshimori, 2001). Antibody dilutions were 1/40 (MN13) and 1/250 (anti-mouse IgM-Alexa488 conjugate; Molecular Probes, Inc., Eugene, OR). To stain PLCζ, samples were washed with PBS and permeabilized with 0.1% (v/v) TX-100 in PBS for 30 min. Samples were then incubated in 1% (v/v) normal goat serum (Life Technologies, Inc., Rockville, MD) for 30 min at RT, and labeled with anti-PLCζ antibodies (1/100 [v/v]) for 1 h at RT. Samples were washed 3× in PBS and labeled with anti-rabbit IgG Alexa488 conjugate (1/250 [v/v]) for 1 h at RT. After washing 3× in PBS, samples were counterstained with 5 μg/ml Hoechst No. 33258 and mounted in fluorescent mounting medium (Dako Corp., Carpinteria, CA). Fluorescence was visualized on a Zeiss Axiovert 200 microscope equipped with a BioRad Radiance 2100 laser scanning confocal system. Images were acquired using LaserSharp 2000 software and image data exported as 8-bit TIFF files and processed using Adobe Photoshop CS. All antibody dilutions were in PBS containing 1% (v/v) normal goat serum.