Hi I am interested in IF staining macrophages in frozen colon sections.
I chose F4/80 as it is a pan-macrophage marker. However, I have hard time staining macrophages in mouse colons.
Typically, I fix freshly excised colons in 4% PFA for 2h at RT followed by sucrose priming (30%) overnight and flash-freezing samples. I think this protocol is pretty standard and it has worked well for staining other antigens.
I have used two biotin-conjugated antibodies for staining F4/80: 1)Thermofisher, 13-4801-82 and 2) BD Biosciences, 565635. Both of them resulted in very weak or almost no staining even at low dilution rate that Datasheet suggests.
If anybody who works a lot with macrophages and routinely stain macrophages on frozen sections, could you please share the protocol?
By the way, I cannot use organic solvent-based fixation (e.g. methanol/acetone) as other antigen I co-stain with F4/80 are sensitive to that. So I can only use aqueous fixatives.
Thanks,
Two reasons could explain your bad result. Duration of fixation is the first one. 2 h is not so much. I would recommand to stay longer in the Fixative, 6 h to over night fixation. The second reason: if you only see single cells and you use Streptavidin-Alexa488, these singel cells could be swallowed by the autofluorescence bachground. In that case I would recommand to change the fluorophores (StreptAvidin -Alexa546 or 647; CY3 or CY5). Possibilities to improve your results:
Enhancement : if you are working with ABC- or Streptavidin -Peroxidase you can use the biotinylated tyramid-technique. These enhancement-technique is also suatable for IF. You can also use the streptavidin-anti-streptavidin-technique or if you are staining you immunos with DAB you can enhance the DAB with Ni and/or Cobald. Instead of a brown reaction product you will recieve a blue/black ones.
Thanks Ute,
I did not think of changing fixation time as this condition has worked for other antigens. I will try fixing longer and see how it goes before touching on other staining techniques. Thanks for your suggestion!
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