CyTOForum

Hi All,

I have optimised a protocol for IMC and I used to get really nice images. Lately, i have been getting many hotspots al over my images that have very high readout and completely throw the images off. I can see the specific staining in the background but it is full of these hotspots that ruins it. i was wondering if anyone else is facing a similar problem and was ablate fix it?

Man thanks,
Sarwah

Hi Sarwah,

Can you attach an image showing the hotspots here so we can get an idea of what you specifically mean? There are usually some hot pixels that appear here and there and they can be removed by either histogram clipping or a median filter.

Best,
Santhosh

Hi Santhosh,

Sorry for the delay in replying, I complexity forgot I posted this here. Please find attached images for p53 and CD45, more visible in p53 image. Do you know how these are formed and if there is something in the staining procedure I can change to reduce them? I currently do not filter my antibodies, do you think filtering the antibody mix would help?

Many thanks,
Sarwah