Toluidine blue

Mast cells were differentiated from hematopoietic cells derived from bone marrow according to the Vukman et al., protocol⁸³ . Cells were cultured for 8 to 10 passages in Iscove’s Modified Dulbecco’s Medium (IMDM) (Thermo Fisher Scientific, 12200069) supplemented with 10% FBS (Thermo Fisher Scientific, 16141002), mrSCF(10 ng/ml) (Biolegend, 579702) and mrIL-3 (10 ng/ml) (Biolegend, 575502). Mast cells were also stained by toluidine blue (Biovitrum, 07-002) — a basic thiazine metachromatic dye that stains nuclei blue and has a high affinity for acidic granules, in accordance with the manufacturer’s recommendations.
According to the flow cytometry analysis of Kit and FcεR1 expression, at the final passages mast cells accounted for ~90% of the total cell population (Supplementary Fig. 3b). For the Hi-C experiment Kit positive cells were magnetically separated (Miltenyi Biotec, 130-097-146).

The animals were sacrificed on days 4, 7, 14 and 21 after the operation, and the wound tissues were excised for histological analysis. Then, 4 μm-thick sections of the formalin-fixed, paraffin-embedded tissue samples were stained with hematoxylin and eosin (H & E) (BioVitrum, St. Petersburg, Russia), picrosirius red (BioVitrum, St. Petersburg, Russia), toluidine blue (BioVitrum, St. Petersburg, Russia) and by picro-Mallory (BioVitrum, St. Petersburg, Russia). A LEICA DM4000 B LED microscope equipped with a LEICA DFC7000 T digital camera running under the LAS V4.8 software (Leica Microsystems, Wetzlar, Germany) was used for the examination of the samples. Sections stained with picrosirius red were examined by polarized light microscopy. The panels were composed of microphotographs of central parts of the wounds for standardized assessment of wound healing.

Four-μm-thick sections of the formalin-fixed and paraffin-embedded brain tissue samples were stained with hematoxylin and eosin (BioVitrum, Moscow, Russia) and 0.1% toluidine blue (BioVitrum, Moscow, Russia) using the Nissle method. Leica DM4000 B LED microscope, equipped with a Leica DFC7000 T digital camera running by the LAS V4.8 software (Leica Microsystems, Wetzlar, Germany) was used for the examination of the sections. The measurements of the damaged areas were made using Leica Application Suite, version 4.9.0 (Leica Microsystems, Wetzlar, Germany). To objectify the results of the histological examination, we used the semi-quantitative analysis (scoring) of slides stained by the Nissle. The signs of cerebral tissue degeneration (pericellular and perivascular edema, hyperchromic shriveled neurons, irregularly shaped neurons, hypochromic neurons, satellitosis, neuronophagy, and pyknotic neurons) in every slide were evaluated with 0 to 3 according to the scoring table (Table A2). The analysis of cortex tissue damage was performed on the rats’ brain parietal lobe at the side of MCA ligation. The areas of ischemic brain alteration were assessed in five random fields at ×200 magnification.