heat retrieval - necessary or not? - his-tagged protein detection (Dec/05/2008 )
Hello, everyone!
I'm going to detect the presence of His-tagged protein in the transfected tissue sections (cryosectioning), and I have plenty of questions as I need to elaborate the most efficient protocol for that. the transfected tissue is hamsters muscle. I have primary mouse anti-His-tag antibodies and the secondary antimouse conjugate with FITC or HRP.
First question is - is the heat retrieval step necessary for this kind of antigen?
And my second question is, probably, stupid.. when you apply the immunostaining of the tissue, can you also stain the same sample by standart methods such as hematein-eosin with the following dehydration of samples, or it is better to visualise the cells by other immunostaining?
May be someone could give me the link to some common protocol on which I could base my manipulations? I really do not want to spoil the samples I've got.
Thank you!
I'm going to detect the presence of His-tagged protein in the transfected tissue sections (cryosectioning), and I have plenty of questions as I need to elaborate the most efficient protocol for that. the transfected tissue is hamsters muscle. I have primary mouse anti-His-tag antibodies and the secondary antimouse conjugate with FITC or HRP.
First question is - is the heat retrieval step necessary for this kind of antigen?
And my second question is, probably, stupid.. when you apply the immunostaining of the tissue, can you also stain the same sample by standart methods such as hematein-eosin with the following dehydration of samples, or it is better to visualise the cells by other immunostaining?
May be someone could give me the link to some common protocol on which I could base my manipulations? I really do not want to spoil the samples I've got.
Thank you!
Q1:as far as I know, heat mediated antigen retrieval is required for intracellular antigens, although we perform short antigen retrievals for our membrane bound antigens as well...
Q2: confusing; are you saying you want to do normal DAB (eg) staining on a tissue, and then do an H&E on it as well? If all you want to do is see the cell structure better against the positively stained tissue, maybe you could just counterstain with Hematoxylin? I might be wrong, haven't worked with his-tagged immunogens but this might be the way to go. Doing an H&E on the same tissue would get messy
Q2: confusing; are you saying you want to do normal DAB (eg) staining on a tissue, and then do an H&E on it as well? If all you want to do is see the cell structure better against the positively stained tissue, maybe you could just counterstain with Hematoxylin? I might be wrong, haven't worked with his-tagged immunogens but this might be the way to go. Doing an H&E on the same tissue would get messy
thank you for the clue.
but I did not actually get what did you mean in the Q2 answer - sorry for being so stupid. all I want to see is the general tissue structure and the particles made by the his-tagged protein (I do not know how it actually should behave, as it is the foreign protein for this tissue), if it is expressed...
What kind of tissue do you want to stain? We use phalliodin staining (stains actin) and it always gives a real nice structure in spleen and liver.