Dear all,
we are running a retinal pigment epithelial cell culture on 6-well transwell plates (Coring). For better cell characterization we need sections for the cells still attached to the membrane.
Membranes are cuttet in squares and embedded in TissueTek, snap frozen in liquid nitrogen, then 10µm cryo-sections are made.
However, after slicing, the membrane starts to curl up and detach from the slides. This happens latest after some washing steps following a standard IHC protocol... sometimes the cells remain at the slides, sometimes not.
Any advice on how to prepare cryo-sections from transwells is appreciated.
What we use: membrane is made of polyester, 0.4-µm pore size, slides are Menzel superfrost plus (Thermo Scientific) or FLEX IHC microscope slides (DAKO).
What we tried so far: fixation of cells with 4%PFA prior of slicing, fixation after slicing, drying the slides at 37°C prior of staining, combinations of all this.
I read some papers recommending vibratome slices. Unfortunately we don't have a vibratome available. Also paraffin sectioning was mentioned. But we are interested in lipids, so this is also not an option.
Thanks.
We are working on the same concept using primary RPE cells. I was able to achieve reasonable success with Millicell 0.45 um 12 mm cell culture inserts. Since the membranes are detached following the cryosections we conducted our stainings on the membrane before embedding it into OCT and sectioning. While signals are significantly diminished by freezing the strong targets are still viable. In order to distinguish between the cells and the membrane deposition of i.e. lipids, we used Calcein AM live to stain. Another approach with reasonable success was using z-stack on the cell monolayer and observation of the orthogonal projection. We are also interested in preserving the good morphology of the RPE cells on the membranes but so far we were only able to investigate depositions of the RPE cells to the membrane and not a good cross-section morphology of the RPE cells. Johnson et al use vibratome on non-embedded membranes https://www.pnas.org/content/108/45/18277 I'm thinking of using laser cutter on non-embedded membranes since vibratome is not available for us.
Please see the paper on Laser capture microdissection:
Article Collection of Epithelial Cells from Rodent Mammary Gland Via...
I just wanted to provide an update. The approach I have used with incubating the membrane with primary and secondary, snap freezing the membranes and then producing cryosections has yielded publishable results. The key to success was to stain the cells with calcein-AM to be able to distinguish between the cells and extracellular deposits. The sections should be made from the membrane size and not the cell side. While the efficiency of the process is not very high and finding well-preserved areas require some efforts overall it is an acceptable method. I usually use Cy5 for target proteins it doesn't produce autofluorescence from the membrane. I also use a visible light scan at the confocal.
Anton Lennikov thank you very much for the update! Currently we are sticking to your recommendation on doing z-stack imaging, but I will keep the other approach in mind as well.
- Badges
- Science topic