Re: Homebrew programmable slide stainer: opinions and suggestions wanted...

"Victor" <microscopevic@xxxxxxxxx> wrote in message
news:f0ba3a7f-f20b-4992-b08c-3684fe165679@xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
On Mar 25, 11:31 pm, "Y" <a...@xxx> wrote:
Hi all,

[snip for brievety]

This is a fascinating idea, although in my experience, for the super
short timed stations, such as with acid alcohol, one aspect to
consider strongly is the "dunk speed". Even with the robotic
stainers, because they're really slow to dunk, you can experience a
"gradient effect" on your specimens, where the bottom of the slide has
sat in the acid alcohol for a relatively significant amount of time
longer than the top of the slide, especially considering the total
time inside the reagent.

You may want to speed up the "dunk" for that station.

Also, is this something that you'll want to make your circuit that
enables this stain type to be enabled and disabled? I cannot fathom
making it run linearly when one of the stations is an acid alcohol
station, but I CAN imagine you inserting a rack, hitting a button, and
at time point # whatever, it speeds up the transfer rate, then slows
back down. This will clearly restrict you to a single rack of this
type unless I'm missing something.

Perhaps it'd be easier to use something like the RA Lamb StainMate for
these special stains? The COT-20 is a great linear stainer (as is
pretty much everyting Medite builds), please keep us up to date.
Perhaps consulting also with histonet would be wise.

Hi Victor,

Thanks for your reply.

The problem of the dunk duration is a very interesting one. I thought on the
time the machine needs for the entire process of transfering a basket from
one station to the next , but I didn't consider the complex issue of the
slide up, the horizontal movement and the slide down movements/times.

Now, I measured a lot of transfering cycles of the COT20 yesteday and yes:
it's slower than those when performing manual staining using slide staining
racks, but not that much slower:

- slide up: on average 3.2s
- horizontal movement: on average 5.1s
- slide down: on average 2.8s
- total: on average 11.2s
Note: drain function off.

That's less than I expected: as I'm already writing some lines of the
staining protocol's batch files, I estimated it some 20s.

I searched the Histonet Listserver to find contributions regarding uneven
(H&E) staining. There were some messages, but those seems to be more related
to other possible causes, mainly problems associated with pH of bluing
solutions, quality of tap water, less than perfect deparaffinization and so
on.

IMHO there are several possible ways to overcome the problem of staining
gradient:

- the use of a very diluted acid alcohol, say 0.1% or even 0.05% 2HCl in
ETOH 70%
-.the use of an acid alcohol containing a less agressive acid. I read 20%
glacial acetic acid in ETOH 80% for 1 min in one of the very few protocols I
could find
- Another way to go would be a progressive hematoxylin and no
differentiation at all.

I guess it wil take some (a lot of?) experiments to find out.

The slide stainer will be used staining one basket at a time only.
It will be used for H&E but for special stains as well AND it will be used
for botanical slides too :-). I'm even considering to use it as a tissue
processor.

Buying another slidestainer is no option, I'm afraid: as I am a private user
I can't afford to pay the usual price for this type of equipment: I bought
the COT20 on Ebay for +/- the price of a slide basket :-).

Have a nice weekend!

Yvan.


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