Sara,
Here is our procedure, years of tweaking (it seems) every time the weather changed! Good Luck!
Janet
Florida Hospital Orlando
________________________________
From: histonet-bounces@lists.utsouthwestern.edu on behalf of Breeden, Sara
Sent: Tue 10/16/2007 8:04 AM
To: histonet@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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