THE HUMAN PROTEIN ATLAS BLOG

Sample preparation – planning is crucial

2016-11-22
Antibody Cell Atlas Immunofluorescence


Christian Gnann by the Tecan's Freedom EVO pipetting robot

To prepare all the images for the Cell Atlas, released on December 4, the cells used are primed for staining and microscopy. Sample preparation is an important step when performing immunofluorescence studies. If wrongly applied it can not only cause unsuccessful detection but also generate misleading information.

The sample preparation performed by the Cell Atlas team includes cell fixation, permeabilization, and immunostaining with primary and secondary antibodies.

– The fixation is the crucial step, and different fixation protocols work better for different sets of proteins, Christian Gnann, a research engineer in the Cell Profiling group explains.

However, in a systematic, proteome-wide effort such as the Cell Atlas, a need exists for a standard fixation protocol that works well for the majority of all proteins independent of subcellular localization. Therefore the group has performed studies on different fixation protocols, and has come to the conclusion that cross-linking is essential, and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.

– This protocol that we use covers all kinds of proteins and works for all the different cell lines we use, Christian Gnann says.

Christian Gnann takes part in all the steps of the production: from cell cultivation to antibody dilution, sample preparation and imaging.

– All our steps are automated which gives us a very reproducible process. To be successful with this large type of project concentration and above all, planning is crucial, he says.

Christian explains how it is vital to design all the steps leading up to the final image already when thawing the cells. However, cells are living material.

– You can never be sure of how the cells will grow, so even if you have everything planned out, you have to be flexible.

With a Bachelor in Pharmaceutical Biochemistry from Germany, Christian Gnann has the perfect background to be a research engineer in the lab, where he actually did his Bachelorīs thesis.

– During my Bachelorīs studies we went to on a field trip to Denmark and Sweden, visited among other places SciLifeLab, where I fell in love with the Cell Profiling group.

So Christian ended up doing his thesis in the group, under the supervision of Peter Thul, where he investigated chromatin binding and Golgi associated proteins. After finishing his studies, he came back to the lab to work as a research engineer.

– I took this job and planned to go back to do a Masters, but my stay in Sweden has become longer than I first thought, mainly because it is such a fun group to work in, I am really enjoying the team work that we have! I would love to stay and see all the exciting new projects coming up, maybe Iīll even do my Master here, Christian concludes.

Read the paper on optimisation of the fixation protocol here >>


Frida Henningson Johnson