FW: [Histonet] AutoStainer XL Tweaking

[Bonner, Janet]

Sara,
Here is our procedure, years of tweaking (it seems) every time the weather changed!  Good Luck!      
                                 Janet     
                                  Florida Hospital Orlando

________________________________

From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Breeden, Sara
Sent: Tue 10/16/2007 8:04 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] AutoStainer XL Tweaking



I'm setting up my Leica AutoStainer XL and would appreciate any help
with tweaking the times.  I based my timing for the Autostainer on my
manual stain times but staining is not crisp and looks washed out.  Docs
say eosin is fine but cellular detail is foggy.  Here are my times:


  YOURS                                                                                        OURS

XYL - 3 at 5 min each     ......................................................    3 at 3 minutes

100% reagent - 2 at 30 seconds each ................................   2 at 30 seconds each

95% reagent - 1 at 30 seconds   ...........................................  1 at 30 seconds    

80% reagent - 1 at 30 seconds   .................................................................      none

DI water wash - 1 minute   ......................................................tap water (connected to faucet) - 1 minute

Hematox (currently using Sigma) - 5 minutes    ...............    hematox (Richard Allen #7211) - 2 minutes

Hematox #2  ...........................................................................    hematox (R-A #7211) - 3 minutes

                   
DI water wash - 1 minute  ....................................................... tap water - 1 minute

Acid Alcohol (1%) - one dip  .................................   1% acid alcohol (bought from Polysciences for control issues)-5 seconds

DI water wash - 1 minute  .....................................................   tap water - 1 minute       

                                            

Ammonia water - 15 seconds   .................................................Richard-Allen Bluing Solution -1 minute

DI water wash - 1 minute  ...........................................................tap water - 1 minute

80% reagent - 1 at 30 seconds .......................................................   none

95% reagent - 1 at 30 seconds .................................................  1 at 30 seconds

Eosin (alcoholic) - 10 seconds  ..............................................   Richard-Allen Eosin #7111(1:1 with 95% EtOH) - 15 seconds

95% reagent - 1 at 30 seconds .................................................  1 at 30 seconds

100% reagent - 2 at 1 minute each  .........................................   2 at 1 minute each

XYL - 3 at 1 minute each ......................................................        1 at 1 minute

  Xylene           ........................................................................        2 at 2:30 minutes   

I cut my acid alcohol time down from 15 seconds to one dip but it's
still taking a lot of hematoxylin off.  Should I try 0.5% acid alcohol?
I'm at a loss so far; different hematox? More time in ammonia water?  If
you use this Leica AutoStainer XL, could you offer some Handy Hints?  I
thank you SO much!



Sally Breeden, HT(ASCP)

NM Dept. of Agriculture

Veterinary Diagnostic Services

PO Box 4700

Albuquerque, NM  87106

505-841-2576



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