We are interested to observe localization of GFP-labeled proteins during the budding process.
To immobilize the yeast cells, you can coat the glass slide surface with concanavalin A or lectin before use.
Hi, it could be useful http://link.springer.com/protocol/10.1385/0-89603-386-4:1
Not immuno-fluorescence right, just fluorescence localization? (getting antibodies in requires much more work!) For S. cerevisiae ConA works best in my experience, but they seem to not attach as well as fission yeast like S. pombe.
Like the others said - Con A is good. For best results, I used MatTek dishes and coated them as follows:
MatTEK dishes are 35mm glass bottom dishes (number 1.5), uncoated, ϒ-irradiated (MatTEK corporation P35G-1.5-14-C)
Prepare stock solution of Concanavalin A 10mg/ml in DDW; filter sterilize.
Prepare 1:10 dilution in sterile DDW.
Place 500µl of 1:10 dilution of Con A on the glass. Incubate 5min r.t.
Wash with sterile water x2
Dry in hood (cover with aluminum to prevent dust).
Coated dishes can be kept dry for 3-4 weeks, so you can prepare dishes in advance.
You then 0.5ml of the yeast in medium at low concentration (so you won't have cells on top one another). let the cells sit for 2-5 minutes to settle. then, very gently, remove the medium with cells who didn't and add, again gently, 2ml of medium and go image.
Another option is on a thin layer of agar (with medium, of course). But you need a special microscope slide with a groove for the agar. You pour the agar and let it cool & solidify for a few minutes, then place the yeast, let them a few minutes to settle then place a cover glass.
Hi Jens,
usually we just put few dropplets of yeast culture inside a Fuorodish or Mattek. The culture should not be to concentrated. Wait 2 or 3 minutes and add your imaging medium. Put the dish directly on the microscope stage.
Most of the cells are fixed to the glass. you can image for 3 hours without any problem. The cells start to swim after few divisions. For longer experiments, the microfluidics system (CellASIC ONIX from Millipore) seems to be nice but very expensive.
Good luck
Virginie
Poly - D -Lysein works for coating too, as does Collagen 4. However I'd add that if you're only interested in cells that are stuck, I'd suggests a wash step in your protocol to remove all the un-adhered cells. We do this with Candida yeast, where we leave them in water to adhere to the slide for 30 mins on a light shaker, then we wash the slide lightly with water to remove unadhered cells. Hope this helps,
regards,
Darren
Hi Jens -
I've done some yeast imaging on agarose pads at CSHL's Yeast Genomics Summer Course. The image/link attached show the basic setup.
We performed our imaging on a fully motorized, upright microscope - which allowed us to:
I was there as "the microscope guy" so I didn't make any of these slides/pads myself.
Best,
Justin
http://www.nature.com/nprot/journal/v8/n6/fig_tab/nprot.2013.066_F2.html
Hello Jens, this is Monica, I was asking the same question. Have you tried the ConA?
Do you want to help us looking at the yeast cells using ConA?
Be careful while functionalising your slides with lectins etc. This will have implications on the cell wall, since the mannans will be linked to the lectins and hence no longer exposed. This may induce some wall remodelling. Something to consider.
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