In Situ ZymographyListed By : Michelle Bendeck
University of Toronto
CategoryGenePathwayDiseaseMember Organization
Laboratory protocolExtracellular matrixNAVBO
In Situ Zymography protocol

1. Cut 8 um cryosections of mouse arterial tissue and place on + charged slides as shown below. This arrangement allows for assay of 2 conditions per slide

2. Keep cut slides at -80 °C until use.

3. Wash slides for 5' in ISZ buffer to remove OCT

4. Outline sections with pap pen so that 2 square coverslips can be used per slide, see below

5. Apply ISZ + desired inhibitors to the sections (400 ul per slide area)

6. Incubate slides overnight in a humidified chamber at 4 °C (17hrs)

7. Melt the 0.5% agarose solution in the microwave
a. keep in a 50 °C water bath until use

8. Prepare the prewarmed agarose solution:
a. Combine in the following order: ISZ buffer, inhibitors, agarose to a final concentration of 0.1%
b. i.e. dilute the 0.5% solution 1:5 according to how much you will need
c. Keep solution warm in the 50 °C water bath

9. Remove slides from the cold room, dump off solution and apply fresh ISZ with 1:10000 Hoescht 33528 + inhibitors

10. Incubate slides in the dark for 10 min at RT

11. During the 10 minute incubation, add DQ gelatin (stock 1mg/ml) to the prewarmed agarose solution to a final concentration of 0.1mg/ml (i.e. dilute 1:10)
a. Keep at 50 °C

12. Dump off the ISZ-Hoescht solution and rinse sections with fresh ISZ + inhibitors

13. Dump off rinse solution, pick up excess solution with a kimwipe

14. Apply 40 ul of Agarose/DQ solution to each side of the slide
a. Coverslip immediately with a 22x22 mm glass coverslips

15. Incubate slides at 37 °C for 30 min

16. Image slides immediately under epifluorescence.




ISZ Incubation buffer

500 ml
10 mM Tris-HCl pH 7.6 0.788 g
150 mM NaCl 4.383 g
5 mM CaCl2.2H20 0.37 g
0.2mM NaN3 0.065 g


0.5% Agarose (w/v) in ISZ Buffer

0.25 g agarose / 50 ml ISZ buffer

Inhibitors

10 mM EDTA
10 mM 1'10 Phenanthroline (from 2.5M stock in MeOH)
100 um GM6001 (stock 2.5mM in DMSO)
1 mM PMSF (stock 200 mM in MeOH)
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