Nb300 144

Human parotid gland tissue cryosections embedded in CRYO-OCT Compound (Tissue-Tek) or cells encapsulated in hydrogels were fixed with 4% (w/v) paraformaldehyde (A11313; Alfa Aesar), permeabilized with 0.2–3% (v/v) Triton® X-100 (A16046; Alfa Aesar), and blocked with 3–10% (v/v) goat serum (5058835; EMD Millipore). Samples were incubated with primary antibodies against basement membrane proteins: laminin (NB300-144; Novus Biologicals, 1:200), collagen IV (NBP1-97716, 1:200; Novus Biologicals), perlecan/HSPG2 (NBP1-05170, 1:200; Novus Biologicals), or β₁ integrin (NB100-63255, 1:200; Novus Biologicals), followed by appropriate secondary antibodies conjugated to Alexa Fluor 488 (A11029; Life Technologies/ThermoFisher), Alexa Fluor 568 (A11011; Life Technologies/ThermoFisher) to tag proteins of interest. Nuclear [4′,6-diamidino-2-phenylindole (DAPI); 40011; Biotium] and filamentous actin (A22287; Life Technologies/ThermoFisher) counterstains were used prior to mounting (P36930; Life Technologies/ThermoFisher), if necessary, and confocal imaging (A1; Nikon Instruments) of specimens.

Aortic and cardiac tissues were fixed for 24 h in 4% formalin solution (BDH Prolabo), and subsequently dehydrated in 60% isopropanol (BDH Prolabo), followed by paraffin‐embedding. Aortic media thickness was measured on orcein‐stained sections of the aorta, which allows for accurate assessment of the inner and outer border of the media layer. Orcein staining was further used to assess elastin content and for counting elastin breaks and elastic laminae. The latter is assessed as the average of 8 measurements across the aortic wall. VSMC phenotype was assessed using immunofluorescent myocardin staining (Sigma, sab4200539), and immunohistochemical staining for α‐smooth muscle actin (α‐SMA, Sigma, F‐3777) and proliferating cell nuclear antigen (PCNA, Biorad, MCA1558F). Cardiac fibrosis was assessed using trichrome Masson staining and cardiac hypertrophy was quantified on the cellular level using an anti‐laminin (Novus Biologicals, nb300‐144) staining. Five images off each mouse were recorded for this measurement in different cross‐sectional regions of the heart, and cross‐sectional area of 20 cardiomyocytes was measured per image (final average of 100 measurements). Microscopic images were acquired with Universal Grap 6.1 software using an Olympus BX4 microscope or Celena S fluorescence microscope and quantified using ImageJ software.