Dq gelatin substrate

The in situ gelatinolytic activity was measured in frozen sections (16-μm thickness) as described previously [27 (link)]. At 24 h following ICH induction, the brains were removed and immediately frozen on dry ice. Fresh sections were incubated with fluorescein-conjugated DQ gelatin substrate (Invitrogen, Carlsbad, CA, USA) for 2 h and subsequently fixed and mounted with VectaShield (Vector Laboratories, Burlingame, CA, USA) medium. In this assay, cleavage of DQ gelatin by MMPs results in green fluorescent products. The images were then captured using a confocal laser scanning microscope (LSM700, Carl Zeiss, Germany).

In situ gelatinolytic activity was measured on frozen sections (16 μm thickness) as described previously [30 (link)]. At 24 h following ICH, brains were removed and immediately frozen on dry ice. Fresh sections were incubated with fluorescein-conjugated DQ gelatin substrate (Invitrogen) for 2 h and fixed and mounted with VectaShield medium. Cleavage of DQ gelatin by MMPs results in green fluorescent products. After gelatinolytic activity had been assessed, some sections were fixed with 4% PFA and incubated with primary antibodies specific to neurons (NeuN) or astrocytes (GFAP; Millipore), followed by Cy3-conjugated secondary antibodies. Images were then captured using confocal laser scanning microscopy.

In situ zymography was performed as described previously [27 (link)]. Tumor tissues (n = 4 per genotype) were drop-fixed in IHC Zinc Fixative (BD Pharmingen) for 24 h at 4 °C, embedded in paraffin, and sectioned at 8 μm. The DQ gelatin substrate (Invitrogen) was incubated with tissue sections as previously described [27 (link)].