Formalin-fixed, paraffin-embedded tissue was cut to 5 μm thick sections via microtome and mounted onto microscope slides (Tissue Tack, Polysciences, Inc., Warrington, PA, USA) then dehydrated overnight at 37°C prior to storage at room temperature. Tissues were stained with Verhoeff Van Gieson (Verhoeff solution for elastin; Van Gieson counterstain for collagen), Movat’s Pentachrome (Alcian Blue for GAG, Saffron for collagen, Resorcin-Fuchsin for elastin, and Woodstain Scarlet-Acid Fuchsin for muscle), or Alcian Blue alone (Polysciences, Inc.), following manufacturer’s protocols. All staining protocols included a hematoxylin counterstain.
For N-glycan and ECM peptide MALDI-IMS studies, FFPE tissues were prepared as previously described [11 (link), 12 (link)]. Chondroitinase imaging preparation is novel to this study. Briefly, a 1 mg/mL Chondroitinase ABC (Sigma Aldrich, St. Louis, MO, USA) solution was prepared in 60mM Ammonium Acetate (pH 8 with sodium hydroxide). Solution was sprayed with a TM Sprayer M3 (HTX Imaging, Chapel Hill, NC, USA) under the following parameters: 15 passes, crisscross pattern, 3.0 mm track spacing, velocity of 1200 mm/min, and a dry time of zero. Before enzymatic digest, four antigen retrieval conditions (20 minutes, 95°C) were studied: 1) EDTA pH 8; 2) Citraconic Buffer pH 3; 3) Tris pH 9; and 4) Double antigen retrieval of Citraconic, wash and desiccate, then Tris.
FFPE aortic valve tissue sections were digested with COLase3 after serial treatment with chondroitinase, PNGaseF (N-Zyme Scientifics), elastase, and for MALDI-IMS experiments. Trypsin (Porcine Pancrease, Sigma Aldrich, St. Louis, MO, USA) was applied during preliminary studies to determine the effects of a single enzyme before COLase3. Spray conditions for all enzymes were as previously published [11 (link), 12 (link)]. Samples were digested in high humidity at 37.5°C for either 2 hours (chondroitinase, PNGaseF) or 5 hours (elastase, COLase3). A 7 mg/mL solution of alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was prepared in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA; for chondroitinase, PNGaseF imaging) or 1.0% TFA (for elastase, COLase3 imaging). CHCA matrix solution prepared for peptide imaging was spiked with a standard of 200 femtomole/L [Glu1]-fibrinopeptide B human (GluFib) (Sigma-Aldrich, St Louis, MO, USA). CHCA automatic spray conditions included: 79°C, 10 psi, 70 μl/min, 1300 velocity, and 14 passes with a 2.5 mm offset. Slides for peptide imaging were rapidly dipped (<1 second) in cold 5mM ammonium phosphate and immediately dried in a desiccator.
For N-glycan and ECM peptide MALDI-IMS studies, FFPE tissues were prepared as previously described [11 (link), 12 (link)]. Chondroitinase imaging preparation is novel to this study. Briefly, a 1 mg/mL Chondroitinase ABC (Sigma Aldrich, St. Louis, MO, USA) solution was prepared in 60mM Ammonium Acetate (pH 8 with sodium hydroxide). Solution was sprayed with a TM Sprayer M3 (HTX Imaging, Chapel Hill, NC, USA) under the following parameters: 15 passes, crisscross pattern, 3.0 mm track spacing, velocity of 1200 mm/min, and a dry time of zero. Before enzymatic digest, four antigen retrieval conditions (20 minutes, 95°C) were studied: 1) EDTA pH 8; 2) Citraconic Buffer pH 3; 3) Tris pH 9; and 4) Double antigen retrieval of Citraconic, wash and desiccate, then Tris.
FFPE aortic valve tissue sections were digested with COLase3 after serial treatment with chondroitinase, PNGaseF (N-Zyme Scientifics), elastase, and for MALDI-IMS experiments. Trypsin (Porcine Pancrease, Sigma Aldrich, St. Louis, MO, USA) was applied during preliminary studies to determine the effects of a single enzyme before COLase3. Spray conditions for all enzymes were as previously published [11 (link), 12 (link)]. Samples were digested in high humidity at 37.5°C for either 2 hours (chondroitinase, PNGaseF) or 5 hours (elastase, COLase3). A 7 mg/mL solution of alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was prepared in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA; for chondroitinase, PNGaseF imaging) or 1.0% TFA (for elastase, COLase3 imaging). CHCA matrix solution prepared for peptide imaging was spiked with a standard of 200 femtomole/L [Glu1]-fibrinopeptide B human (GluFib) (Sigma-Aldrich, St Louis, MO, USA). CHCA automatic spray conditions included: 79°C, 10 psi, 70 μl/min, 1300 velocity, and 14 passes with a 2.5 mm offset. Slides for peptide imaging were rapidly dipped (<1 second) in cold 5mM ammonium phosphate and immediately dried in a desiccator.
