Histostar workstation

After fixation, kidney tissues were dehydrated by applying gradually increasing ethanol concentrations in the Excelsior MS tissue processor (Thermo Fisher Scientific, Waltham, USA). Next, the tissues were embedded in paraffin with the HistoStar Workstation (Thermo Fisher Scientific) and 5 µm thick tissue sections were made using with Microm HM 355S microtome (Thermo Fisher Scientific). Tissue sections were stained with the Periodic Acid Schiff’s (PAS) staining kit (Carl Roth GmbH, Karlsruhe, Germany). Histological assessment was performed with a Leica DM 2000 microscope. The percentage of collapsed glomeruli and renal blood vessels with intravascular accumulation of one or more leukocytes was calculated on whole sections.

Testes were collected from C57BL/6 (WT), Plcz1^−/− and Plcz1^−/−eCS^−/− male mice (N ≥ 3 each) after euthanasia and then fixed with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) overnight at 4 °C. Fixed testes were dehydrated through an ethanol gradient and embedded in the paraffin using the Cell & Tissue Processor CT-Pro20 (GenoStaff, Tokyo, Japan) and the HistoStar workstation (Thermo Fisher Scientific, Waltham, MA, USA). Paraffin sections measuring 6 µm were prepared on an RX-860 microtome (Yamato Kohki Industrial, Saitama, Japan) and stained with hematoxylin and eosin (H&E) according to the standard procedures for histological evaluation. Spermatogonia, spermatocytes, round spermatids, and elongating/elongated spermatids were evaluated morphologically. Photomicrographs were taken with a BZ-X700 microscope (Keyence, Osaka, Japan). The representative image is shown.

Lungs from each mouse were fixed in 10% neutral buffered formalin (Soluform^tm, JLS-Chemical, Russia) at +4°C, dehydrated in isoprepe (BioVitrum, Russia) using Microm STP 120 (Thermo Scientific, USA), and embedded in HISTOMIX (BioVitrum, Russia) using HistoStar workstation (Thermo Scientific, USA). 5-μm thick sections (10 per mouse) were cut using a Finesse ME+ microtome (Thermo Scientific, USA), stained with haematoxylin and eosin and mounted in Vitrogel (all BioVitrum, Russia). Pictures were obtained either using an Epson Perfection V600 scanner (9600 dpi) or using an Imager. Z1 microscope with an AxioCam MRc 5 camera (all ZEISS, Germany) at 20х and 63х magnification. Pathological changes were assessed using three parameters: (1) the acute lung injury (ALI) score, showing mostly the immunopathology in the lung parenchyma (from 0 to 1); (2) the peribronchiolar infiltration score, estimating the degree of inflammation of the tissue around the bronchioles (from 0 to 5); and (3) the perivascular infiltration score, estimating the degree of inflammation of the tissue around vessels (from 0 to 5). The exact parameters of tissue scoring systems are provided in the Supplementary Materials (p. 9).