I have done this staining before and did not have any issue. The negative control which did not have a primary antibody looks good.
By lines, are you referring to the rod-shaped things?
The dots look like contamination. Do you spin down your primary and secondary antibody dilutions before adding to the sample?
Also, it would be helpful to explain in your images what is the specific and what is the non-specific signal.
Hello,
Your images are an assemblage of images (mosaic) with stitching issue, explaining the lines you observe.
The illumination is slightly different for each image (different background, different intensity signal) leading to these lines.
Hi Stacy, I did not spin down my primary and secondary ab dilutions. I will spin them next time to see if it helps.
Yes, I am referring to the rod-shaped things as lines.
The non-specific staining are the dots in the first image and the rod shape things in the second image.
Nicolas Barois I added a separate image of the red channel. The lines are from the red channel.
Likely to be aggregates or contamination. You could just dilute your secondary Ab in buffer, put on a slide and see if they are there. You probably need new secondary Ab or aliquot.
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