Glu1 fibrinopeptide b human glufib

The formalin-fixed, paraffin-embedded (FFPE) tissues and TMA were prepared as previously described.^{[19 (link)]} Briefly, tissues were heated, dewaxed, antigen retrieved at pH 9, and sprayed with a thin molecular layer of collagenase type III (COLase3; StemCell Technologies) using an automated sprayer (M3 TM-Sprayer, HTXImaging, Chapel Hill, NC, USA). COLAse3 was sprayed onto tissues with the same automated sprayer using parameters of 45°C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes with a 3.0 mm offset. Samples were digested in high humidity at 37.5°C for five hours followed automated spraying of CHCA matrix prepared as 7 mg/mL in 50% acetonitrile, 1% TFA with a spiked standard of 200 femtomole/microliter [Glu1]-Fibrinopeptide B human (Glufib) (Sigma-Aldrich), St. Louis, MO, USA). CHCA was sprayed onto tissues with the same automated sprayer using parameters of 77°C, 10 psi, 100 μL/min, 1300 velocity, and 10 passes with a 2.5 mm offset.

The formalin-fixed, paraffin-embedded (FFPE) tissues and TMA were prepared as previously described.[18 (link), 19 ] Briefly, tissues were heated, dewaxed, antigen retrieved at pH 9 using 10 mM tris buffer, and sprayed with COLase3 using an automated sprayer (M3 TM-Sprayer, HTXImaging, Chapel Hill, NC, USA). COLAse3 was sprayed onto tissues using parameters of 45°C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes with a 3.0 mm offset. Samples were digested in high humidity at 37.5°C for five hours followed by automated spraying of CHCA matrix prepared as 7 mg/mL in 50% acetonitrile, 1% TFA with a spiked standard of 200 femtomole/microliter [Glu1]-Fibrinopeptide B human (Glufib) (Sigma-Aldrich), St. Louis, MO, USA). CHCA was applied by automated sprayer with parameters of 77°C, 10 psi, 100 μL/min, 1300 velocity, and 10 passes with a 2.5 mm offset. Slides were rapidly dipped (<1 s) in cold 5 mM ammonium phosphate, monobasic and dried in a desiccator prior to imaging mass spectrometry data acquisition.