Aligned digestion spots were chosen on six serial sections of both fresh frozen and fixed mouse liver and mouse caudate putamen slides (each 10 μm thick). First, five cycles of hyaluronidase enzyme solution (0.16 TRU/μL hyaluronidase in the presence of 2 M ammonium acetate and 10% glycerol) was applied on the chosen spots. The resulting HA disaccharides were extracted four times by 0.3% ammonium hydroxide solution and the slides were dried at 55 °C for 5 min, which was followed by 20 min incubation at 37 °C with 50 mM ammonium bicarbonate. Next, five cycles of chondroitinase ABC (1 milliunits/μL) enzyme solution was added. To minimize the amount of salt in the enzyme solution, the 5 mM Tris buffer was replaced by 25 mM ammonium bicarbonate buffer and 10% glycerol was also added to minimize diffusion. The resulting CS disaccharides were extracted as the HA disaccharides and the slide was dried at 55 °C for 5 min, which was followed by 20 min incubation with 25 mM ammonium bicarbonate at 37 °C. Next, five cycles of heparin lyase I, II and III mixture was added. The solution contained 1.66 mU/μL of heparin lyase I, 0.33 mU/μL of herparin lyase II and 0.33 mU/μL of heparin lyase III in the presence of 2.5 mM calcium hydroxide, 12.5 mM ammonium bicarbonate and 10% glycerol. The resulting HS disaccharides were extracted as the HA and CS disaccharides and the slide was dried at 55 °C for 5 min, which was followed by 20 min incubation with 50 mM ammonium bicarbonate at 37 °C. Next, a solution containing 10 mM DTT, 0.1% RapiGest and 10% glycerol was applied on the chosen spots and the tissue slide was incubated at 55 °C for 20 min. This was followed by addition of a solution containing 20 mM iodoacetamide, 25 mM ammonium bicarbonate and 10% glycerol and the tissue slide was placed in a darkbox for 20 min at room temperature. Next, five cycles of trypsin enzyme solution (100 ng/μL) in the presence of 10% glycerol were added to achieve digestion of proteins. Samples were then incubated with aprotinin (2 μg/μL) at 37 °C for 45 min to stop trypsin activity. Finally, five cycles of PNGase F (500,000 units/mL) were added. The peptides and glycans were extracted by 10% acetic acid and separated by C18 spin column. Samples were loaded in 5% ACN/0.1%FA, the flow-through and wash fraction contained the N-glycans, while peptides were released first by 40% ACN 0.1% FA then by 60% ACN 0.1% FA, combined and dried under vacuum. Individual cycles consisted of microwave irradiation for 10 min (for CS, HS and proteins, 270 W; for HA, 540 W) except for N-glycans where incubation took place in an incubator (37 °C, 40 min/cycle). Subsequent LC-MS methods and data interpretation can be found in the Supporting Information along with a table summarizing the experiments performed (Table S-1, Supporting Information).