Hello, everybody,
a student of ours wants to show cell wall changes (thickenings) in ultra-thin sections of Arabidopsis (LR White). To detect callose, the sections were stained with 0,01 % aniline blue in 150 mM K2HPO4 (pH 9.0) for 15 min in the dark and rinsed with water. Unfortunately, the chloroplasts and plasma membranes also stain unspecifically, even with shorter incubation times.
Does anyone have any advice on how we can minimize the background?
Thank you very much and many greetings from Hamburg!
Judith
Dear Dr. Mehrmann, just to clarify (honestly):
your student will NOT stain ultrathin sections with the Anilin-blue in K2HPO4 at pH9.0, but tries to stain semithin sections (~0.50 - 1 µm - 2 µm- thick sections) to locate the expected callose-substrate in the sections and do then correlative EM using the positive staining result -as seen by LM - to choose the right area for 'ultrathins'? Right?
Thank you for clarification....
Dear Dr. Muss,
I´m just a TA, but thanks for the Dr.!
You're right, of course, she is using semi-thin sections (1-2 µm). She is trying to repeat an experiment from a former pHD student, using a confocal laser microskope.
Dear Ms/Mrs Mehrmann, apologies (:-)) for treating you with a "Dr." title. As I am an informal "retired - hence dispensable or unimportant - person" I thank you - in return - also for the "Dr."(which I am) but I also am happy with my first name [wenn Sie mich damit ansprechen wollen...gerne!.(:-)) ]
I've gone through my e-library and have done a google-RG-search too and would likely have answered with my thoughts (but perhaps by mailing your institutional address you might have to my private e-mail-address [email protected]. That would be better for the ease of further communication, a resumee I naturally would post afterwards here in this particular thread).
Meanwhile or in parallel I would like to encourage you to give some information on the former experiment of the PhD-Student-colleague (at least basic parameters of techniques/methods used...). It may well be that other or somehow modified
staining methods will do the trick and yield better or positive results....
Thank you for your confidence....(admitting that I do have some kind, friendly and
- towards me - well disposed RG-colleagues at the UKE in Hamburg...:)
Best regards and greetings to the "Waterkant", Wolfgang H.M.
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