I haven't done this exact thing before but I was able to do immunofluorescence analysis on cells incorporated in low melting point agarose. The biggest issue with the technique is the detachment of the agarose layer from the microscope slide but can be done if you are careful. Also, everything needs to be ice-cold, this helps with the detachment problem.

Mateo Glumac i actually do not want to detach the cells from the coverslip... as I am looking at cell migration and cell-substrate interactions. I want to overlay the cell sheet with agarose and then perform immunofluorescence staining while the whole thing (even the explant) is still attached to the glass coverslip. Do antibodies permeate through agarose? How difficult is it to completely wash out non-binding primary and secondary antibody from the gel?

Hi Alekhya,

Have you tried pre-coating the microscope slides with either poly-lysine, collagen or similar before incubating with the explant? I'm not sure what your experiments are so consider whether this would interfere with your fluorescence target or other downstream analysis you might have planned. I have done this to attach endothelial and smooth muscle cells to microscope slides and it didn't interfere with our immunofluorescence.

Alekhya Hati Sorry, my answer was not clear enough. Yes, antibodies permeate through agarose without any problem.

You should pre-coat the microscopic slide with 1 % standard agarose by dipping it in and removing the excess agarose from the opposite side of the slide.

Then use low melting point agarose that melts under 40 °C (so that you don't cook the cells) and add another layer of agarose on top of the preexisting agarose layer and push in the coverslips containing cells facing up (still attached to the coverslip). After the agarose solidified, add another layer of LMP agarose to cover the cover-slips. You can try fixing cells before and after the addition of the agarose layer to see if there is any difference. I would do cell permeabilization after incorporation into agarose to prevent the loss of cells that you are experiencing.

Agarose is very permeable and any excess antibody should be easily removed by doing 3-5 washouts with PBS-Tween 0.05%, at least this was the case with my experiment.

I hope that my advice was helpful and that your experiment succeeds.