Hello cell slave,
thanks so much for your help!
Your totally right, I have to work on using the microscope.
Yes, I have added immersion oil on the slide.
I will try to clean everything and improve the pictures.
Thanks for all your suggestions, I will definitely try out some of them.
The major problem is, that I do not work with cell culture.
For chromosome preparation, I dissect male ants, isolate their testis und pull testis into pieces to release cells (everything is happening on the slide already).
I incubate in colchicine solution to stop meiosis in metaphase.
KCl isn´t included in my protocol, but instead a hypotonic solution of Na3Citrat x 2H2O
Do you think the kind of hypotonic solution makes a difference?
Consequently, I do not drop anything on the slide, as testis are so tiny I would definitely lose them in a tube.
Nevertheless, I will try storing slides in -20°C Methanol, as you suggest.
Humidity is a good point as well, it´s also mentioned in the protocol I use, I will try that as well.
I am not sure about 2 things you mentioned: mitogen and thymidin:
I do not use any mitogen, as cells in male testis are dividing anyway.
Do you think I should add it to the protocol?
As far as I know thymidin is inhibiting DNA synthesis, right?
As I already use colchicine to arrest in metaphase, I do not know if I should try?
After colchicine treatment, I use three different fixatives stepwise: 2 different Ethanol-GAA mixtures and finally GAA purely.
Than I apply a standard Giemsa stain.
So you think it´s not a problem with the stain but with the chromosome preparation, right?
I am very grateful for your help!
I know that want I´m doing is totally different from human cell culture, but I will try out your suggestions!