High overlap between CD8 CD68 CD3

[Katitamorais]

Hello!

We are running a cycle combining CD8 CD68 CD3. We got good fluorescence signal and specificity using FFPE. However, when we used fixed frozen tissue (1:4 Cytofix/Cytoperm Fixed Frozen), there is a high overlap between CD8 CD68 CD3.

CD3 - clone EPR4426 - validated for IBEX FPPE -https://www.abcam.com/en-us/products/primary-antibodies/alexa-fluor-555-cd3d-antibody-ep4426-ab208514
CD68 - clone KP1 - validated for IBEX FFPE and Cytofix/Cytoperm - https://www.capricobio.com/product/cd68-kp1-ifluor-594/
CD8 - clone C8/144B - validated for FFPE - https://www.biolegend.com/de-de/products/alexa-fluor-647-anti-human-cd8a-antibody-14127

Interestingly, when CD8 and CD4 were evaluated together in fixed frozen tissue, we could see specificity.

Now we will run a test simultaneously evaluating 4 tissues types and both FFPE and fixed frozen tissue.

Have you already seen anything similar?

Thanks.
Kátia.

[radtkea]

Hi Katia,

Thank you so much for asking your question in the open! Apologies for my delay in answering. You can always @radtkea to get me directly.

Thank you for providing the images and clones you are using.

My answer: you need to use different clones/antibodies for the fixed frozen tissues. There are a few clones (CD68 clone KP1) that work in both fixed frozen and FFPE tissues. You can search for appropriate clones in the reagent resources table by selecting "1:4 Cytofix/Cytoperm Fixed Frozen" in the tissue preservation method tab. You rightly noted this in your answer above!

For CD4, I recommend the RPA-T4 clone. It's available in many different conjugates from different vendors. AF647 is a great choice.

For CD3, I recommend the UCHT1 clone. It's also available in many different conjugates from different vendors. iF594 was used throughout our studies.

For CD8, I recommend the SK1 clone. I used it mostly in AF488 and used a 1:25-1:40 dilution.

I have not used the CD3 and CD8 clones you specified for fixed frozen tissue. As to why they might work together "separately" but not together is spectral overlap: the CD68 iF594 might be spilling into the AF555 and AF647 channels. This is the one clone (KP1) validated for both tissue preservation methods. If you titrate it, then you might be able to see the CD3 and CD8 better.

I would avoid PE in the colon. It appears to bind non-specifically in gut tissue.

Best of luck! Please reach out if you need anything else.
Andrea