Gelatin Zymography Can Be Performed on Fixed Brain Tissue

Abstract

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

Keywords: brain; gelatinase; matrix metalloproteinases; tissue fixation; zymography.

Figure 1.

ISZ with the MMP fluorogenic substrate DQ-gelatin in the peri-contusional cortex of cryosections. 10 min of ethanol, acetone, zinc or PFA fixation didn’ t inhibit gelatinolytic activity (A, B, C, D, E); The broad-spectrum MMP inhibitors 1,10-phenanthroline (K, L, M, N, O) and EDTA (metal ions chelating agent) (P, Q, R, S, T), but not PIC (a protease inhibitor cocktail) (F, G, H, I, J), abrogated gelatinolytic activity. Representative figures from three independent experiments. Scale bar=100 µm. Abbreviations:DQ, dye-quenched; ISZ, In situ zymography; PFA, paraformaldehyde; MMP, matrix metalloproteinases.

Figure 2.

With the extension of the PFA fixation time (A: 30 min, B: 1hour, C: 2 hours; D: 6 hours, E: 12 hours, F: 1 day, G: 3 days, H: 7 days), the gelatinolytic activity gradually weakened. Representative figures of 3 independent experiments. Scale bar = 50 µm. Abbreviations: ISZ, In situ zymography; PFA, paraformaldehyde.

Figure 3.

Protein extraction efficiency: (A, B) Triton and NaCl as well as DMSO and NaCl were ineffective at extracting proteins from ethanol-, acetone-, ZBF-, and PFA-fixed brain tissues. (C) the concentration of protein extracted by urea and CHAPS was low in ethanol- or acetone-fixed tissues, and even lower in ZBF-, and PFA-fixed tissues than in fresh tissues. (D) SDS could extract proteins from unfixed tissues and ethanol-, acetone-, and ZBF-fixed tissues but not from tissues fixed with PFA for 24 hr. (E) SDS with heating could extract proteins from all the unfixed tissues and ethanol-, acetone-, ZBF-, and PFA-fixed tissues. (F) the protein extraction efficiency of SDS was gradually reduced with the extension of the PFA-fixed time. All n=6. Abbreviations: CHAPS, 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate; DMSO, dimethyl sulfoxide; PFA, paraformaldehyde; SDS, sodium dodecyl sulfate; ZBF, zinc-based fixative. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

Gelatinase extraction using 8 M urea and 4% CHAPS: (A) gel silver staining analysis revealed that the molecular weight distribution of extracted proteins from ZBF-fixed tissues was completely different from that of fresh tissues and ethanol-, and acetone-fixed tissues, and the extracted proteins from PFA-fixed tissue were mostly low-molecular-weight (less than 20 KDa) proteins (green box). (B) WB revealed that MMP-9 and MMP-2 can be effectively extracted from fresh tissues and ethanol-, acetone-, and ZBF-fixed tissues but not PFA-fixed tissue. (C) IGZ revealed that gelatinolytic activity was preserved in the fresh tissue extraction as well as ethanol-, acetone-, and ZBF-fixed tissue extractions, but not PFA-fixed tissue extraction. Conditioned media of HT1080 cells served as a positive control. Representative figures from three independent experiments. Abbreviations: CHAPS, 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate; IGZ, in-gel zymography; MMP, matrix metalloproteinases; PFA, paraformaldehyde; WB, western blotting; ZBF, zinc-based fixative.

Figure 5.

Gelatinase extraction using 2% SDS: (A) gel silver staining analysis revealed that the extractions from fresh tissues and ethanol-, acetone-, and ZBF-fixed tissues contained proteins of various molecular weights, except for those from PFA-fixed tissue, which contained mostly those low-molecular-weight proteins (green box). (B) WB showed that proteins of MMP-9 and MMP-2 can be extracted from all the fresh as well as ethanol-, acetone- or ZBF-fixed tissues but not from PFA-fixed tissues. (C) IGZ revealed that the gelatinolytic activity was preserved in fresh and ethanol-, acetone-, and ZBF-fixed tissue extractions, but not in PFA-fixed tissue extraction. Active MMP-9 was better preserved in ethanol- and acetone-fixed tissue extractions (red box). Conditioned media of HT1080 cells served as a positive control. Representative figures from 3 independent experiments. Abbreviations: IGZ, in-gel zymography; MMP, matrix metalloproteinases; PFA, paraformaldehyde; SDS, sodium dodecyl sulfate; WB, western blotting; ZBF, zinc-based fixative.

Figure 6.

Gelatinase extraction using 2% SDS with heating: (A) gel silver staining analysis revealed that proteins were extracted using 2% SDS with heating from all the fresh as well as ethanol-, acetone-, ZBF- and PFA-fixed tissues. (B) WB revealed that proteins of MMP-9 and MMP-2, as well as β-actin, were extracted from all the fresh as well as ethanol-, acetone-, ZBF, and PFA-fixed tissues. (C) IGZ revealed that gelatinolytic activity was completely abrogated in fresh and ethanol-, acetone-, ZBF-, and PFA-fixed tissue extractions. Conditioned media of HT1080 cells served as a positive control. Representative figures from three independent experiments. Abbreviations: IGZ, in-gel zymography; MMP, matrix metalloproteinases; PFA, paraformaldehyde; SDS, sodium dodecyl sulfate; WB, western blotting; ZBF, zinc-based fixative.

Figure 7.

Effect of PFA fixation time on gelatinase extracted using 2% SDS. (A) gel silver staining analysis revealed that as the fixation time was increased, macromolecular proteins became more difficult to extract. (B) WB revealed that the extraction efficiency of MMP-9 and MMP-2 proteins decreased as the PFA fixation time increased. (C) IGZ confirmed that gelatinolytic activity in the extractions gradually decreased as the PFA fixation time increased. Conditioned media of HT1080 cells served as a positive control. Representative figures from three independent experiments. Abbreviations: IGZ, in-gel zymography; MMP, matrix metalloproteinases; PFA, paraformaldehyde; SDS, sodium dodecyl sulfate; WB, western blotting; ZBF, zinc-based fixative.

Figure A1.

Segmentation and gelatinase expression of TBI brain tissues. (A) The brain tissues were sliced into 1 × 1 × 1 mm³ pieces, and those from different regions were divided into 5 equal portions. (B) IGZ confirmed that the contents of MMP-9 and MMP-2 were equivalent in those 5 portions. Conditioned media of HT1080 cells served as the positive control. Representative figures from three independent experiments. Abbreviations: IGZ, in-gel zymography; MMP, matrix metalloproteinases; TBI, traumatic brain injury.