Stp 120

The dissected tissue samples were fixed in 4% PFA (Wako) for overnight at room temperature. The next day, the samples were poured into 70% ethanol (Wako) for several hours, and then poured into 80% ethanol, 90% ethanol, 100% ethanol, and xylene (Wako), and embedded in paraffin using a spin tissue processor (STP120; Thermo Scientific). Sections were sliced into 3–4 μm thickness, and stained with hematoxylin and eosin (Muto Pure Chemicals). Serial sections were used for the immunohistochemical analyses.

The samples were fixed by 4% PFA at 4°C overnight and embedded into paraffin (Paraplast, Sigma‐Aldrich) by tissue processor (Cat. #STP 120, Thermo Fisher Scientific). Then the rotation microtome (Cat. #HM325, Thermo Fisher Scientific) was used to cut paraffin sections (4 µm). The sections were incubated with indicated primary antibody and FITC‐labelled secondary antibody in humidified atmosphere (antibodies listed in Table S5). Nuclei were labelled with DAPI (2 μg/mL). The results were photographed by fluorescence microscope.

Rat kidneys were freshly fixed in 4% PFA, washed in RNase free water and transferred in 70% RNase free ethanol. Kidneys were bisected longitudinal before automated embedding in paraffin using a STP120 (Thermo Fisher Scientific). Each paraffin-embedded half was sectioned (10-μm sections) using a microtome HM340E (Thermo Fisher Scientific).
Histologic staining was performed on deparaffinized and hydrated serial sections of rat kidney. Hematoxylin and eosin (H&E) staining visualized cell nuclei (black, dark blue) and counterstains cytoplasm and connective tissue fibers (different shades of pink). In detail (also shown in Table 1), staining was started by deparaffinization with two steps of absolute xylene followed by rehydration steps with a descending ethanol row. Hematoxylin staining was done with Mayer’s hematoxylin solution (Carl Roth GmbH, T865.1) for 10 min followed by 10 min bluing in lukewarm running tap water. Counterstain was done with 1% Eosin Y solution (Carl Roth GmbH, 3137.2) for 2 min followed by a differentiation step in 70% ethanol for 30 s. Stained sections were mounted with Roti-Histokitt (Carl Roth GmbH, 6638.1). Stained sections were stored at room temperature until imaging analysis was performed.

The bladder tissue samples were fixed by 4% PFA containing 2% sucrose in PBS at 4°C for overnight and embedded into paraffin (Paraplast, Sigma-Aldrich Ltd., USA) using a tissue processor (Cat. #STP 120, Thermo Fisher Scientific Ltd., UK). Paraffin sections (4 μm) were cut with a rotation microtome (Cat. #HM325, Thermo Fisher Scientific Ltd., Germany). The sections were serially incubated with primary antibody and Cy3-labeled or FITC- labeled secondary antibody in humidified atmosphere (Tables 3 and 4). Nuclei were labeled with DAPI (2 μg/ml). Immunofluorescence staining images for paraffin sections were analyzed by the fluorescence microscope.

Wound tissues were removed, fixed in 10% phosphate-buffered formalin, and processed automatically using the STP-120 (Thermo-Fisher Scientific, Waltham, MA, USA) histology station. Paraffin-embedded tissues were cut by a microtome, i.e., НМ340Е (Microm Laborgerate GmbH, Berlin, Germany). Four-mm-thick frontal sections were stained by hematoxylin and eosin and viewed under a light microscope, OLYMPUS BX51 (OLYMPUS, Tokyo, Japan). We also used Van Geison’s staining to assess collagen production. The severity of the inflammatory reaction, number of fibroblasts and fat cells, and level of epithelization were examined using the Shekhter et al. histological score table [32 (link)].

In hamsters, tissue samples were taken from the large right lobe of the liver and (i) were placed into RNAlater (ThermoFisher Scientific, Waltham, MA, USA); (ii) were fixed in a 10% aqueous solution of neutral formalin and were dehydrated in a graded series of ethanol and in xylene (STP-120, Thermo Scientific). Dehydrated samples were enclosed in a paraffinic medium HISTOMIX (BioVitrum, Saint Petersburg, Russia). For microscopic examination, sections of 3.5 μm thickness were prepared on a rotary microtome Microm HM 355S (ThermoFisher Scientific, Waltham, MA, USA). Human gallbladder tissue samples along with liver samples were subjected to similar chemical processing.

Liver samples from the experimental and control groups were processed, cleaned, and placed in 0.9% (w/v) NaCl physiological solution for 30 min, then fixed in 10% formaldehyde solution for 24 h. The processed tissues were dehydrated in increasing concentrations of 70%, 80%, 90%, 96% absolute alcohol, and finally with xylol for 1 h for the substitution of alcohol by xylol. Then the tissues were submerged in molten paraffin (62 °C) for the xylol to come out of the tissue and be replaced by paraffin. Finally, cubic molds containing the tissue were filled with paraffin, and after cooling, they were cut with the aid of a sliding microtome (rotary hand processor STP 120, Thermo Scientific MICROM, International GmbH (Robert-Bosch-Str. 49 69,190 Walldorf/Germany) in sections in serial layers with a thickness of 4 mm. These were extended by floating on the surface of warm water. Subsequently, the slides were immersed in descending concentrations of xylol and alcohol for tissue staining by the hematoxylin method and eosin (H and E). This process was performed in triplicate to obtain adequate image resolution of the tissue. Finally, examination was conducted through the light electron microscope at 20X [11 (link),23 (link),38 (link)].