Phosphotungstic acid haematoxylin (PTAH) staining for fibrin

Using this stain, fibrin appears dark blue while nuclei, cytoplasms, red blood cells and fibrils appear pale blue or purple. Collagen takes on a red tone. The protocol ⁴⁴ followed was: 1. Incubate slide in 50% Lugol's iodine until a yellow-brown colour is acquired, 2. Eliminate excess iodine and wash in 0.5% potassium thiosulphate solution, 3. Wash in distilled water for 5 min, 4. Incubate slide in 5% sodium thiosulphate for 3 min, 5. Rinse in distilled water, 6. Incubate in an oxalate solution for 1 min, 7. Rinse in distilled water, 8. Stain with PTAH for 2 h at 60 °C, 9. Dehydrate: 2 × 1 min passes in 96% alcohol and 2 × 1 min passes in 100% alcohol, 10. Clear sections in xylol for 10 min. 11. Slides were mounted with Cytoseal™.

For transmission electron microscopy(TEM), small fragments of vein tissue were introduced into 3% glutaraldehyde for 1-2 h. Next, the fixed samples were washed in Millonig buffer for at least 2 h. Embedding was done over three consecutive days. Semi-thin sections of 1 μm thickness were cut with an Ultracut Reichert-Jung (Reichert Technologies, Depew, NY, USA) ultramicrotome and then stained with Toluidine blue and visualized under a light microscope. Once the area of interest was identified, the blocks were carved with a shaper (Creighert-TM 60), and ultra-thin sections of 60 nm prepared. The sections were collected on copper grids coated with a Formvar resin membrane and treated with lead citrate for 4 min. Once washed and dried, the sections were visualized with a tabletop TM microscope (TM-100).