Mounting methods

Relaxing stiff specimens fixed in 100% ethanol

Most effective approach for relaxation of stiff specimens preserved in alcohol or formalin is incubation with enzymes

Method 1. Proteinase K (expensive!).

Chemicals from DNeasy Blood & Tissue Kit

Things needed:

• 1. Eppendorf tube

  2. Buffer ATL (to be stored in fridge)

  3. Proteinase K (to be stored in fridge)

1) Check Buffer ATL. Clear at 55°C if precipitated. Use heating cabinet.

2) Set incubator to 55°C.

3) Add 180 μl ATL

4) Add 20 μl proteinase K

5) gently shake tube using Vortex machine

6) Incubate at 55° C for 24 hours (should be vortexed a couple of times)

Specimens soft like after ethyl acetate and ready for mounting

Method 2 with pepsin (cheap)

Things needed:

• 1. Eppendorf tube

  2. Pepsin

  3. HCL

  4. Distilled water

1) Prepare solution: 1% pepsin, 1% hydrochloric acid (served as buffer) and 98% distilled water.

2) Add Pepsin solution and specimens to Eppendorf tube

2) Incubate at 37°C for 24 hours.

If specimens remain stiff, continue incubation for more 24 hours.

Notice

• • Relaxation time of large or very stiff (after formalin) specimens may be longer.

  • Do not put in the tube big amount of specimens it may prevent or inhibit the chemical processes.

  • Do not use enzymes at to low or to high temperatures.