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
Hi Mike
If you red is more Like Brown it is well known- you Need a new Set.
Greetings
Gerhard
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
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
Dear Gerhard, thank you!
If you don't mind, can I trouble you for your process?
Obrigado!
Mike
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
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
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