Lead citrate

After fixation of arterial samples in 2.5% glutaraldehyde (TED PELLA, CA, USA) in PBS (pH 7.2), specimens were post-fixed in 1% osmium tetroxide (Heraeus, Hanau, Germany), dehydrated in graded ethanol and propylene oxide (Acros Organics, USA), and then embedded in Epoxy resin (mix with Nadic Methyl Anhydride (NMA) and Dodecenyl Succinic Anhydride (DDSA) and DMP-30, all reagents from Polysciences (PA, USA). Serial ultrathin sections were cut using an LKB-III ultratome (LEICA, Wetzlar, Germany). Ultrathin sections were stained with uranyl acetate (TED PELLA, CA, USA) and lead citrate (TED PELLA, CA, USA) and were examined with the aid of a Hitachi H7600 electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV.

Sections (70 nm) of the central retina were cut from resin-embedded samples, placed on copper grids and counterstained with 2% uranyl acetate and 3.5% lead citrate (Ted Pella, Redding, CA, USA). The samples were imaged on a JEM-1400 electron microscope (JEOL, Peabody, MA, USA) at 60 kV with a digital camera (BioSprint; AMT, Woburn, MA, USA). Image analysis and processing were performed with ImageJ.

For the electron microscopy assay, semen samples were prepared in accordance with a protocol previously described [19 (link)]. Briefly, for scanning electron microscopy (SEM), samples were sequentially dehydrated using an ascending gradient of ethanol (Shengqiang Medical Technology, Jiangsu, China) and then dried with hexamethyldisilane (HMDS, Sigma-Aldrich, Castle Hill, NSW, Australia). Samples were then air-dried, added dropwise to specimen stubs, sputter coated, and examined using field emission SEM (Nova Nano 450, Thermo Fisher Scientific Inc., USA). For transmission electron microscopy (TEM), samples were fixed with 2.5% osmium tetroxide (Sigma-Aldrich, Castle Hill, NSW, Australia) and sequentially dehydrated using graded ethanol (50, 70, 90, and 100%) and 100% acetone (Sigma-Aldrich, Castle Hill, NSW, Australia). Samples were then infiltrated with acetone and SPI-Chem resin and embedded with Epon 812 (SPI#02659-AB, Structure Probe, USA). Subsequently, samples were sliced using an ultra-microtome (UC7, LEICA EM, Germany) and stained with uranyl acetate (#19481, Ted Pella, Inc., Redding, CA) and lead citrate (#19312, Ted Pella, Inc., Redding, CA). Cryoelectron microscopy (TecnaiG2 Spirit 120 kV, FEI, Netherlands) was used for image capturing.

A498 cells were harvested in 1 mm³ blocks and immersed in 3% glutaraldehyde solution for 2 h. The samples were post-fixed in 1% osmium tetroxide (OsO₄; CAT#: 18,459; Ted Pella, Redding, CA, USA) for 2 h, dehydrated in ascending alcohols, and then embedded in Eponate 12 resin (CAT#: 18,005; Ted Pella, Redding, CA, USA). Ultrathin (60 nm) sections were cut and post-stained with 5% uranyl acetate (CAT#: 19,481; Ted Pella, Redding, CA, USA) and lead citrate (CAT#: 19,312; Ted Pella, Redding, CA, USA). The mitochondrial ultrastructure of A498 cells was observed under a transmission electron microscope (JEM 1011, Japan).

HE staining: Paraffin-fixed blocks were serially cut into 5-6 μm-thick coronal sections. For routine histological examination, the paraffin sections were stained with HE. HE-stained slices were analyzed under a Nikon upright microscope (Nikon Eclipseci, Tokyo, Japan). 10 fields of view (100 ×) were randomly selected from each group to measure the thickness of the periarterial lymphatic sheath in each field (Mingmei Optical Fiber Digital Measurement and Analysis System V1.5.3) for statistical analysis. Three slices were selected from each group and 15 fields (400 ×) were randomly captured to calculate the number of megakaryocyte precursor cells in each field of view, and performe statistical analysis.
TEM: Each spleen was divided into small blocks of 1 mm three and then fixed with 2.5% glutaraldehyde at 4°C. Ultrathin slices with a thickness of 50–70 nm were prepared and stained with uranyl acetate (22,400, EMS, USA) 30 min and lead citrate (19,314, TED PELLA, USA) 15 min The samples were examined under a transmission electron microscope (JEM-1400, Tokyo, Japan). 20 fields of view (2,500 ×) were randomly selected from each group to calculate the total number of cells, the number of apoptotic cells in each field, and proportion of cell apoptosis, then performe statistical analysis.