Has anybody experience with WGA staining in formalin-fixed and paraffin-embedded tissue sections?
I've been trying to visualize the cell membrane in mouse heart sections, but so far I only managed to get a weak signal.
I would be very grateful for every input. Thank you everybody!
Kind Regards,
Edward
Hi Edward, what kind of WGA did you use? biotinylated or fluorophore labelled?
Sometimes it helps to increase the concentration of the WGA or to play with the detection systems. For examople if you use the biotinylated WGA you can detect either by Streptavidin-Fluophor or Streptavidin-POX-DAB. I would recommand the latter one, because you can better distinguish between real signal and autofluorescence.
Thank you for your answer, Ute!
I used fluorophore labelled WGA and I will now try to increase the concentration.
Kind regards,
Edward
HI Edward. We've been using WGA on fixed tissue culture cells. I will say that one sticking point for us was the use of HEPES buffer. Without it, we saw no staining. Are you using HEPES buffer for your WGA staining step?
Hi Michael. Thank you for your input! So far we have not been using HEPES buffer for the WGA staining. Can I ask you at which steps you use the HEPES buffer?
Edward. According to the manual from Thermo, the WGA should be diluted in HBSS for staining. Afterwards, you can stain with additional antibodies. See the attached link. I would assume your product would have a similar protocol, but check with the manufacturer. Good luck.
https://tools.thermofisher.com/content/sfs/manuals/mp00831.pdf
Thank you, Michael! I will now try to dilute the WGA with HBSS instead of PBS.
Kind regards,
Edward
Hello Ashraf, we used the fluorophore labelled WGA because it was already available in our lab at that moment. Do you have better experience with AP or HRP conjugated WGA?
I use WGA Alexa Fluor 488 Conjugate on PFA fixxed and paraffin-embedded sections, and it works pretty well.
It is diluted in PBS for a final concentration of 5 micrograms/mL.
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