Fig 2: Morphological equivalents of mast cell CPA3 secretory pathways. Primary antibodies used: Rabbit polyclonal to CPA3 antibody (ab251696) (a–u) (AbCam), and Mouse monoclonal [NK1/C3] to CD63 antibody (ab1318) (f) (AbCam). Secondary antibodies used: (a–c,g–l,n–q,s,t) AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP), SDT GmbH, Baesweiler, Germany, label-HRP. (d–f,m,r,u) Goat anti-rabbit IgG Ab (#A-11034) Invitrogen, label-Cy3. (f) Goat anti-mouse IgG Ab (#A-11029), Invitrogen, label Alexa Fluor 488. (a,b) Tonsil. Compound CPA3 exocytosis with probable involvement of piecemeal degranulation. Selective accumulation of CPA3 in targets of a specific tissue microenvironment (indicated by an arrow) (a), accumulation of protease in the pericellular space of the extracellular matrix (indicated by an arrow) (b). (c) Tonsil. Formation of MC loci with the most intense CPA3 secretion (indicated by an arrow). (d,e) Skin. Secretion of CPA3 into the extracellular matrix by the mechanism of compound exocytosis and piecemeal degranulation, with the accumulation of protease in the pericellular space of neighboring cells (indicated by an arrow) (d). Formation of a separate MC cytoplasmic locus for CPA3 secretion (indicated by an arrow) (e). (f) Melanoma of the skin. High content of exosomes in CPA3+ mast cell. (g,r) Morphological variants of targeted degranulation of CPA3 by the mechanism of exocytosis to cellular targets in the specific tissue microenvironment of the tonsilla (g–j) and skin (k–m) (indicated by an arrow). (n–p) Tonsil. The initial stages of mast cell degranulation by the mechanism of exocytosis (n,o) (indicated by an arrow), leading to the distribution of CPA3 + secretory granules in the extracellular matrix (p) (indicated by an arrow). (q–r) Skin. Active exocytosis of mature secretory MC granules into the extracellular matrix with the formation of loci of the tissue microenvironment with a high content of CPA3+ granules (indicated by an arrow). (s–u) skin process of formation of CPA3+ macrovesicles of small (s) and large sizes (t,u) (indicated by an arrow). Scale bar: 5 µm.

Fig 3: Representative double fluorescence immunostainings in the adult SCN. GFAP (green) used as an astrocyte marker, GR (red) and DAPI (blue) as a nuclear marker. Brain was collected from a 90-day old mouse at ZT (Zeitgeber time) 7, immediately frozen on dry ice, and sectioned (12 µm) in a cryostat. After fixation (20 min at room temperature (RT)), sections were blocked with goat serum 4% in phosphate buffer (PBS) containing 0.4% of Triton X-100 for 1 h at RT. Sections were incubated with anti-GR (Abcam Ab183127, 1:200 in blocking) for 2 nights at 4°C. Sections were incubated afterwards with anti-GFAP (ThermoFisher 14–9,892-82, 1:200 in blocking) for 1 night at 4°C. The day after, sections were incubated with secondary antibodies – goat anti-rabbit Alexa 594 (ThermoFisher A11012, 1:500) and goat anti-mouse Alexa 488 (ThermoFisher A11029, 1:500) in a dark chamber for 2 h at RT. DAPI staining was performed to stain nuclei by incubating slides with 300 nM DAPI (ThermoFisher D1306) in PBS for 2 min in the dark. Images of immunofluorescent staining were made using Nikon Ts2R-FL (10X (insets) and 40X magnification).