W3ASim II - shelf life / Lagerfähigkeit ?

[Michael Cytrynowicz]

Dear forum members - as always, please forgive me for not writing in German (I am learning, though)

10 years ago Fahrenheit very generously sent me (back then, in the USA, California) - vials with W3ASim II, and the 3 separate stains as well. He also sent me a vial of Etzold FCA.

I've been starting again to make botanical cuts, and while the Etzold seems perfectly good, I am not getting good results with the WRASim II (the 6:4:1:1 prep).

What I get is VERY weak red coloring, and nothing else. I have been following the staining protocol carefully, staining 5 min at 60 C, differentiating, etc.

Could it be that the 6:4:4:1 solution is expired?

I will try the W3A (original) next, with the 3 Farben separately, but just wanted to know if anyone has experience with related issues.

Many Thanks!

Mike / Portugal

[Rawfoto]

Hi Mike

If you red is more Like Brown it is well known- you Need a new Set.

Greetings

Gerhard

[Michael Cytrynowicz]

Dear Gerhard,

thank you!

Do all 3 stains expire, or is it more the Acridin Red?
But, I am looking at the photos I made during the process, and it seems that at some point a lot of the red got washed out.

My process has been W3ASimII > 2x Aqua dest 1 min > 3x Isopropyl 99%, at 30 s, 3 min, 5 min

Could the Isopropyl, or the Aqua dest washes be an issue?

Many thanks!

Mike

[Rawfoto]

Good Morning Mike

I am using the 3 Color stand alone, but this is Not the problem.

Aqua and ISO are washing out, but should Not be the problem. I am using Etanol in between for washing out, 30% und 70%, 70% is the critical step!

Jörg Shared a Tipp with us, one drop of the Color on a tisue should Show the 3 different Color if they are dry.

Greetings
Gerhard

[Michael Cytrynowicz]

Dear Gerhard, thank you!

If you don't mind, can I trouble you for your process?

Obrigado!

Mike

[Michael Cytrynowicz]

Good Afternoon Gerhard,

More specifically - I am confused, the protocols I have for W3ASim II have the following steps:

Fix (usually AFE) > Ethanol 70% > etOH 50% > etOH 30% > dAqua 3x > Stain > dAqua 2x > ISO 3x > Euparal

So are you saying there needs to be 2 more Ethanol steps, after staining?

Like this:

Fix (usually AFE) > Ethanol 70% > etOH 50% > etOH 30% > dAqua 3x > Stain > dAqua 2x > etOH 30% > etOH 70% > ISO 3x > Euparal

Thank you!

Mike

[Rawfoto]

Hi Mike

I am controlling the washing via checking with the help of my microscope (Wild M3C). For this I am using the 2 steps with Ethalol, it is mainly the 70%. It is possible to skip the 2 steps, but the cells are more stressed if you Go direct to 99%

Greetings

Gerhard

[Michael Cytrynowicz]

Ah, thank you Gerhard,

So after staining, wash in water 2x, wash in etanol 70% 2x, then to Isopropil 99% right?

And to go back to the original problem, which was that W3ASimII, the colors were fading dramatically, so the etanol 70% prevents the colors from fading, is that what it does?

What I am curious, is why the available protocols don't mention these etanol steps.

Warm Regards,

Mike