Dear Jaya,

this depends a bit on what you want to stain and how you want to evaluate your data.

But basically, the answer is yes. You can stain consecutive sections with different markers and then register the images onto each other. There are several tools for this, e.g. elastix (https://elastix.lumc.nl/). A colleague of mine is working on this subject and he implemented elastix into an ImageJ macro (Fiji). You can find the code here: https://github.com/jo-mueller/Slice2Slice.

Since microcopy images are usually quite big, it is recommendable to do a pre-evaluation before the registration to down-sample the resolution and make the process faster. For example, using binary images that only contain two information: marker-positive and marker-negative. This can also be done with Fiji (thresholding, trainable segmentation) or e.g. with Illastik (trainable segmentation).

But please keep in mind that these are not the exact same cells! Even if you do very thin paraffin sections (2 - 3 µm are possible), and more so if you perform cryofixation and have thicker slices.

Hope this could help.

Best,

Theresa

Dear Jaya, have you considered to perform the first staining round, image acquiring and afterwards boiling the samples to remove the antibody and fluorochromes and re-do the staining with your second set of antibodies? Then you can ensure to have the exact same cell stained. As Theresa mentioned there are several software solutions to register the images and merge them into one with multiple channels.

Best

Lukas

Thank you all for the answers. Will try with your suggestions.