I am desperately looking for a good primary that can be used to detect vessels in rodent lung tissue by IF. I have previously tried vWF and CD31, which both resulted in high background staining. Does anyone have any experience with isolectin IB4 in the lung? Does it pick up endothelial cells only?
After searching for a suitable antibody online, most antibodies that I was able to find were for use in human tissue. For the one's that I did find, there were no IF sample pictures, as they had been used for WB.
Currently, I am struggling with the specificity of the antibodies that I have used so far, as they also pick up the alveoli and/or have a high non-specific staining due to the samples being paraffin-embedded and PFA fixed.
Any recommendations and/or tips and tricks on how to reduce the background staining in paraffin-embedded slides would be very much appreciated.
Thank you!
DAKO has a very good polyclonal for VWF staining: A0082. It works for PFA fixed samples and both in human and mouse. In the brain I get very nice/clean staining of the endothelium.
The fluorescence you are seeing is due to the fact that your tissues are FFPE (formalin-fixed, paraffin-embedded), which greatly increases tissue autofluorescence. It is not antibody-related; changing the antibodies or the target will not change this autofluorescence. For FFPE tissues it is much more preferable to use chromogenic staining techniques (eg. DAB IHC) than fluorescence. If you have to use fluorescence for some reason, you'll need to use microscopy techniques that allow to 'gate out' the fluorescence, like confocal microscopy.
- Badges
- Science topic