Pathy staining

From:Patricia Bourne

1.  Slides should be soaked in buffer at least five (5) minutes before staining.

2.  The machine may be level but the "sink" which holds the racks may not.  Put one of the little round levels on a slide in position #6 which is the most important one.  This will tell you if the sink is level. 

3.  We put little square pieces of "rubberish" shelf liner under the feet of each machine to keep it from move as the arm moves.  This will also help keep the machine in the same place.

4.  To check the drop zones, place hematoxylin in a vial and program the machine using just this reagent.  You can place it in the first position as a primary.  Place slides in each slide position and put buffer on each slide.  Next start a "fake" run and watch where the drops are landing and how they spread. You can mark each slide on the slide grid and see if a pattern forms.  Occasionally, the pattern may not fit the optimal spread.  I usually confirm this with a couple of trails just to make sure of the pattern I am getting.  If you find several slides that don't look good, then call the Dako rep to come in and adjust the pattern.  Each machine may have a different pattern so it is important to test both machines.

5.  We add an additional 1.0 ml of tween 20 to each 10 liter of buffer to help with the spread of the antibodies.

6.  If you use the machines a lot, your probe may need to be changed.  We usually see things starting to slide after about six (6) months.

7.  Make sure your buffer and distilled water lines are not growing anything.  We had bad water and found our lines were full of bacterial growth.  We now change our lines at least once every six (6) months or more if necessary.

8.  You can also take some of the same cases you see pathy staining and the increase the reagent amount ex.  if you now put on 100 microliters per drop zone you can increase it to 150 per zone and let the slides go through a complete run and compair your slides and see if the staining is better.  If the staining is better then you can tell it is a machine problem. 

9.  We have found that fresh xylene is very important.  We change all our xylene containers each week. 

Hope this helps.  Let me know how much of this you have done and maybe I can help more.  Good luck!




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