Hi All,
I would like to know if there is a way to superimpose serial sections of tissues after doing immunofluorescence. The problem is I want to see the co-localization between few proteins and the antibodies are either mouse or rabbit. (No other species available). So I can only add two antibodies at a time. I was thinking if it would be possible to do the IF on serial tissue sections and superimpose on each other like the way people do for cryoEM. Any input or help would be appreciated.
Thanks
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
