Q: Fixation Agents for Microscopy

[cmos]

Hey all,

Let me start off by saying that I am not a biologist, hence the ignorance of my questions!

Basically, I'm wondering what are some common fixation agents used in optical microscopy? Specifically, I'm interested in looking at unstained epithelial cells. The most "advanced" methods I use are phase contrast, dark field, and oil immersion; so nothing too special in terms of imaging method. The only caveat (in case it is of any concern) is that, at times, I have to place the cells on a hydrophobic substrate.

Any other comments (e.g. specific preparation methods) would be appreciated. Thanks in advance!

 

[mack_10]

cell cytologists often just use heat

smear cells on slide very thin then dry by passing through bunsen burner, I don't think you will see much unstained.

what are you hoping to see?

 

[Andy Resnick]

Hey all,

Let me start off by saying that I am not a biologist, hence the ignorance of my questions!

Basically, I'm wondering what are some common fixation agents used in optical microscopy? Specifically, I'm interested in looking at unstained epithelial cells. The most "advanced" methods I use are phase contrast, dark field, and oil immersion; so nothing too special in terms of imaging method. The only caveat (in case it is of any concern) is that, at times, I have to place the cells on a hydrophobic substrate.

Any other comments (e.g. specific preparation methods) would be appreciated. Thanks in advance!

I generally use 4% paraformaldehyde for the initial fixation. There's also (cold) methanol and glutaraldehyde methods.

http://en.wikipedia.org/wiki/Fixation_(histology)

Fixation will not increase the contrast- fixation simply keeps the cell (and its contents) locked in place so that a stain may then be applied. The chemicals used are not that safe; from the tone of your question I wonder if you have the equipment to handle the materials safely.

Phase contrast should give quite acceptable images of unstained cells- I use cheek swabs in demos- but if you are having trouble, you can try DIC (differential interference contrast) imaging or one of the variants like Hoffman modulation contrast.

 

[mack_10]

"Let me start off by saying that I am not a biologist"

please don't start using formaldehyde or glutaraldehyde without expert help and safety gear esp a fume cupboard and gloves

 

[cmos]

Thanks to everybody for the comments and especially for the concerns. I work in a materials lab, so we're should be good on the safety end. At the very least, we're usually able to determine necessary safety protocols when bringing in a new process.

In a nutshell, we're trying out some side ideas that have come up along the way in regards to microscopy. Really, all I'm trying to figure out is this: once I prepare a sample (e.g. cells in "something" between a slide and a cover slip) is there a way to keep it in place such that I can go back and look at it several hours later?

From what I've been seeing, even if a fixation agent gives you a longer time period (with respect to water) to work with, it will still degrade the sample. The problem I've been having with water is that it seems to evaporate too fast; I assume due to part to my use of a hydrophobic substrate. In regards the drying the sample, we'd like to keep it aqueous, or at least in a fluid.

 

[Andy Resnick]

From what I've been seeing, even if a fixation agent gives you a longer time period (with respect to water) to work with, it will still degrade the sample. The problem I've been having with water is that it seems to evaporate too fast; I assume due to part to my use of a hydrophobic substrate. In regards the drying the sample, we'd like to keep it aqueous, or at least in a fluid.

Whoa- hang on there. When I fix in PFA, I wash the PFA away after 20 minutes. After wash steps, staining, etc, the cells are mounted in Vectashield, which is a chemical preservative. The specific Vectashield I use has DAPI in it to counterstain the nucleus.

http://www.vectorlabs.com/catalog.aspx?catID=279

How are your growing your cells? If you grow them on a coverslip-especially epithelial cells- you can fix them, mount them, and attach the coverslip to a slide for viewing. Keeping that in a refrigerator will result in a slide that can last for years.

I strongly urge you to locate some simple protocols for fixing (and staining) cells first. Here's an outline of what I do (epithelial cell cultures): feel free to PM me for additional detail, etc.

1) When cells are ready to be fixed, add 4% PFA in Dubelcco's Phosphate-buffered saline (DPBS) for 20 minutes at room temperature (RT)
2) Remove most PFA
3) wash 3x with DPBS (3 min/wash)at RT
4) Incubate in quenching buffer (75mM NH4Cl, 20 mM Glycine in DPBS) for 10 minutes at RT.
5) Wash 3x with DPBS (3 min/wash) at RT
- stop point- (can put cultures in refrigerator overnight, if needed)
6) permeabilization step- you may omit and skip directly to step 13
7) wash 3x DPBS
8) Blocking step
9) incubate with primary antibodies
10) wash 3x
11) incubate with secondary antibodies
12) wash 3x
-stopping point- (can put cultures in refrigerator overnight, if needed)
13) remove all fluid, add a drop of Vectashield to cells
14) attach a coverslip, seal edges with nail polish

A few notes-
NEVER wash or use plain water in solutions- you will lyse the cells.
Even though the PFA will fuse the cells to the substrate, use caution when washing/changing fluids so as not to dislodge the cells.
The quench buffer is important- don't skip that step.
Vectashield needs to be stored at 4 C, or it will degrade.