Fluoroshield with dapi histology mounting medium

Cells were cultured on a coated 13 mm glass coverslip (PDL and laminin, as aforementioned) for 48 h in a complete embryonic NeuroCult™ proliferation medium. The cells were washed with PBS, fixed with 4% paraformaldehyde (MERCK #30525-89-4) in PBS for 20 min at room temperature (RT), and subsequently permeabilized with PBS containing 0.5% Triton X-100 for 5 min. Then, the cells were incubated with a blocking solution containing 10% goat serum and 0.2% BSA (Biological Industries Israel Beit Haemek #030-010-1B) in PBS with 0.1% Triton X-100, for 1 h at RT, followed by primary antibody incubation with anti-Nestin (anti-rat 1:250, abcam ab81462) at 4 °C overnight. Next, secondary antibody donkey anti-rat 488 (1:500, abcam ab150153) was incubated for 1 h at RT. Washing with 0.1% Triton X-100 in PBS preceded each step. Finally, coverslips were mounted in Fluoroshield™ Histology Mounting Medium with DAPI (Sigma-Aldrich #F6057). The representative images were taken with a Nikon Eclipse Ti2 wide-field fluorescence microscope (Fig. 1C).

Isolated monocytes containing platelets were spotted on Alcian blue‐coated glass slides and fixed in cold 4% paraformaldehyde (4°C, 30 min). After fixation, cells were washed with PBS and then blocked in PBS with 0.2% BSA (for surface staining) or PBS with 0.1% Triton and 0.2% BSA (T‐PBS; for intracellular staining) for 30 min at room temperature shaking. Following blocking, monocytes and platelets were incubated with primary specific antibodies against Annexin A1 (AnxA1; 5 μg/ml; clone 1B, in house generated) and Gelsolin (GSN; 1.54 μg/ml clone EPR1942; Abcam, Cambridge, UK; Cat#ab109014, RRID:AB_10863643) in either PBS+0.2% BSA or T‐PBS+0.2% BSA overnight at 4°C. Cells were washed and incubated with secondary antibody Alexa Fluor 488 anti‐rabbit (5 μg/ml, Molecular Probes Invitrogen, Eugene, USA; Cat#A‐11008, RRID:AB_143165) or Alexa Fluor 594 anti‐mouse (5 μg/ml, Molecular Probes Invitrogen; Cat# A‐11032, RRID:AB_2534091) in T‐PBS+0.2% BSA for 1 h at 20°C shaking. Cells were then mounted with a glass coverslip using Fluoroshield Histology Mounting Medium with DAPI (Sigma‐Aldrich) and visualized under the microscope Zeiss LSM800 Imaging System.

For each data point, a minimum of 500 sperm per straw were assessed using previously described methods (Lieberman et al., 2016 (link)). Briefly, frozen semen straws were thawed in a water bath set at 35 °C for 30 s. Semen was then transferred to a 1.7-mL microcentrifuge tube and incubated at 35 °C for 1 h. Twenty microliters of semen solution was spread on a microscope slides and air-dried for 15 min. The slides were fixed by immersion in absolute methanol for 15 min. Slides were rinsed in PBS baths twice for 5 min each, transferred to a bath containing 25 μg/mL PNA-FITC (Sigma, St. Louise, MO) for 30 min, and rinsed in three phosphate buffered saline baths for 5 min each. Slides were gently dried using compressed air. One drop of Fluoroshield with DAPI Histology Mounting Medium (Sigma, St. Louise, MO) was then applied to each slide and acrosome integrity was measured using fluorescence microscopy.