Dm 6b light microscope

Turbidity, zeta potential and concentration of orthophosphates were measured in supernatants and membrane filtrates after TRTs following standard procedures [37 ].
Samples of supernatants were filtered before turbidity and zeta potential measurements through the quantitative cellulose filter paper with the pore size 8–12 µm (Grade MN 640 md, Macherey-Nagel™, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany).
Zetasizer Nano-Z (Malvern, Malvern, UK) was used for zeta potential measurements. HACH 2100 N IS Turbidimeter was used for turbidity measurements (ISO 7027 method). The concentration of orthophosphates was measured using EasyChem Plus colourimetric analyser (SysteaTM, Systea S.p.A., Anagni, Italy) by following the automated method (USEPA Method 365.1).
Samples of sediments were tested for Capillary Suction Time (CST) using T304 test cell (Triton Ltd, Essex, UK). Microscopic images of sediments were taken at ×90 magnification with Leica DM 6B light microscope (equipped with Leica DMC4500 camera) and used to identify particle size distribution (PSD). Area of 2544 × 1816 pix was analysed for each image using ImageJ software package, identifying particle areas and converting into diameters that were used for the cumulative distribution plots according to the [38 ]. The PSD results are expressed according to the [39 ] as distribution points, mean values, span and uniformity.

The tissue sections (7 μm thick), after deparaffination and rehydration, were incubated in 3% H₂O₂ for 20 min at room temperature (RT). After blocking the non-specific sites in 1.5% normal goat serum (NGS) diluted with phosphate-buffered saline (PBS) pH 7.2 (NGS cod S1000, Vector Laboratories, Burlingame, CA, USA), sections were incubated with rabbit polyclonal anti-GPR30 antibody (dil. 1:500, cod. PA5-28647, Invitrogen, Waltham, MA, USA), mouse monoclonal anti-ESR1 (dil. 1:200, cod. TA807239, OriGene Technologies Inc., Rockville, MD, USA), and rabbit polyclonal anti-ER-beta (dil. 1:500, cod. PA1-311, Invitrogen, Waltham, MA, USA) overnight at 4 °C.
After washing in PBS, the sections were incubated with HRP-conjugated secondary antibody (dil. 1:4, cod. UNIHRP-050 ImmunoReagents, Raleigh, NC, USA) for 30 min at RT. The immunoreactions were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (SK-4100, Vector Laboratories, Burlingame, CA, USA). In addition, the sections were counterstained by using hematoxylin. The negative controls included omission of primary antibody (Supplementary Materials).
The immunoreactions were observed by two different blind observers who then captured images and analyzed them using a Leica DM 6B light microscope and SFC7000T digital camera.

Frozen healthy aorta sections were cut at 7 µm and stained with hematoxylin (Mayer, Clin-Tech Limited, Guildford, United Kingdom) and eosin (in house). For the visualization of Lp(a), tissues were fixed with acetone (Sigma-Aldrich) for 10 minutes, followed by 10 minutes of blocking with Ultravision Protein Block (Thermo Fisher Scientific). The tissue was stained with apo(a) (LPA4, 1:25) for 1 hour, washed, and incubated with the secondary antibody (poly-HRP-anti-mouse IgG, undiluted; ImmunoLogic, Arnhem, NL) for 30 minutes. Apo(a) was visualized with DAB (3,3’diaminobenzidine; ImmunoLogic) and counterstained with hematoxylin. Sections were imaged on a Leica DM6B light microscope.