Re: Automated EM processing; problems with lynx.

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From:Jeff Crews <>,
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This is our current protocol:

VIAL #          SOLUTION                             RUN TIME

1          0.1M Caco. Buffer                         15 mins.**
2          0.1M Caco. Buffer                         15 mins.
3          0.1M Caco. Buffer                         15 mins.
4          1% 0s04 or 0.2% RuO4 in Caco Buffer       2 hours**
5          0.1M Caco. Buffer                            15 mins.
6                      H2O                              15 mins
7                      H2O                              15 mins.
8                     2% Uranyl Acetate                  1 hour.
9                     H2O                               15 mins  
10                    H2O                               15 mins. 
11                    H2O                               15 mins. 
12                     50% EtOH                         15 mins. 
13                    70% EtOH                         15 mins.  
14                    95% EtOH                         15 min
15                     100% EtOH                       15mins.**
16                    100% EtOH                        15 mins.
17                     Propylene Oxide                 30 mins.
18                     Prop. Oxide/Epoxy 1: 1         24 hours** 
I hope that this is legible and that the formatting doesn't get 
screwed up in the mail. As you can see, it's much the same as 
yours except for the osmication, en-bloc uranium staining, and 
the rinses that those steps require. However, we do allow more 
infiltration time in the 1:1. The "24 hours" is programmed in so 
that it will sit overnight. After that we change the stem and 
baskets into new 1:1 for the rest of the day, and then put it in 
straight epoxy (actually, we are using a mix of Embed 812 and 
araldite) under vacuum for a couple of hours before embedding. 
We generally use flat molds because orientation is usually 
pretty important to us in the skin business.   
        We are using the shallow baskets that occupy one space 
between the holes on the stem. We could fit more than 4 on the 
stem, but we never do due to fears of having insufficient 
reagent:tissue ratios. Four shallow baskets has always given us 
good results and we haven't needed ot exceed that.
I would consider how much reagent you are using in relation to 
the amount of tissue. Also, does the tissue have plenty of room 
to move around in the basket? I assume that you are using about 
1mm cubes of tissue. 
If you are using the hinged circular cover that fits around the 
basket stem and covers the reagent vial, make sure that you 
don't squeeze it shut so far that it grips the stem. If you do, 
it won't agitate: the arm will move up and down but the stem and 
baskets will not. And finally, make sure that your 100% is 
stored over Molecular Sieves to keep it dry. I always fill the 
100% and PO vials last.
        Our protocol may be overkill, especially with the 
multiple changes of epoxy, but the extra time and reagents are 
insignificant compared to the cost of reprocessing or losing a 
sample. Drop me a line if I was unclear on any of this.

Jeffrey S. Crews, HTL (ASCP)
Organogenesis, Inc.

Thankyou for your reply. Our processing schedule is as follows; 
Pot 1.  30% Alcohol - 15 mins
Pot 2.  50% Alcohol - 15mins
Pot 3.  70% Alcohol - 15 mins.  Pot 4. 90% Alcohol - 15 mins. 
Pot5.  100% Alcohol - 15mins.  Pot 6. 100% Alcohol - 15mins 
Pot 7. 100% Alcohol - 15 mins. Pot 8. 100% Alcohol - 15 mins. 
Pot 9. Propylene Oxide - 10 Mins . Pot 10. Prop.Oxide 10 mins. 
Pot 11. Prop.Oxide - 10 mins.
Pot 12. 50:50 Prop. Oxide/Resin - 60 mins
Pot 13. Resin - 60 mins.  Pot 14. Resin - 60 mins. 
Pot 15. Resin - 60 mins.
Total processing time is 7.5 hours.
We think that our main problem is with air bubbles created by the dipping of 
the arm on the processing machine. Could you please let us know which size 
basket you use as you mentioned that you can get 4 baskets on the stem.  The 
baskets that we use are the deeper ones and we can only get three on the 
stem. We seemed to have more problems with the smaller shallower baskets.
Look forward to hearing from you at

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