Perform under hood.

Do not use this procedure with tissue that has been stained with fluorescent dye.

Will need:

250 mL DI water

250 mL 50% ethanol (50% 200 proof ethanol: 50% part DI water)

250 mL 75% ethanol (75% 200 proof ethanol: 25% part DI water)

250 mL 95% ethanol (95% 200 proof ethanol: 5% part DI water)

500 mL 100% ethanol (200 proof)

500 mL citrosolv

1. Immerse slides (in slide boat with metal handle) in DI water for 30 sec.
2. Immerse slides in 50% ethanol for 5 min.
3. Immerse slides in 75% ethanol for 5 min.
4. Immerse slides in 95% ethanol for 5 min.
5. Immerse slides in 100% ethanol for 5 min.
6. Immerse slides in 100% ethanol for 5 min.
7. Immerse slides in citrosolv (100%) for 5 min.
8. Immerse slides in citrosolv (100%) for 5 min.

Slides are ready to mount

Important: Prepare solution under the ventilation hood, always wear gloves, mask, and goggles!

1 liter of 0.1M PB (pH 7.35)

40 grams of paraformaldehyde

1. 40 g paraformaldehyde (PF)
2. Place a 1 or 2 L Erlenmeyer flask on a stirring hotplate (in hood!), with a stir bar.
3. Pour the 40 g of PF into the flask + 500 mL of 0.1 PB, and begin stir (the other 500 mL will be added later).
4. On a holding bar (attached to the hotplate), place a thermometer and place into the solution to obtain continuous readings of temperature in the  solution.
5. Gradually start heating.
6. Wait for the temperature to rise, do not allow to rise above 60 degrees. If temperature reaches 60 degrees, and turning down the heat knob does not slow the increase, then use some of the remaining 500 ml 0.1 M PB solution to cool the solution.
7. After the temperature in the solution reaches ~60 degrees (and stays constant), the solution should begin to change from a cloudy to a clear solution.
8. After it clears, continue to stir at 60 degrees for an addition 5-10 min.
9. Now, you must filter the solution before use.
10. Filter.
11. pH the filtered solution to 7.35.
12. Store in +4 degrees, allow it to cool, before using it.

Developer solution= 1 part concentrated developer solution + 4 parts DI water (e.g. 50 mL developer + 200 mL water)

Fixer solution= 1 part concentrated fixer solution + 4 parts DI water (e.g. 50 mL fixer + 200 mL water)

After exposing autorad to ECL-enhanced membranes incubate the autorad in the following solutions, in a flat open container/tub:

1. Incubate autorad in developer solution for 2 min
2. Incubate/rinse in DI water for 30 sec
3. Incubate autorad in fixer solution for 2 min
4. Incubate/rinse in DI water for 30 sec
5. Hang-up to airdry

Parts:

Concentrated developer, from Fisher, Cat. # 05-728-104 (Kodak part number 190-0943)

Concentrated fixer, from Fisher, Cat. # 05-728-112 (Kodak part number 190-1875)