Feb 22, 2019

Public workspaceGlutaraldehyde fixation

DOI
dx.doi.org/10.17504/protocols.io.ycgfstw
  • 1EPFL - EPF Lausanne
Veronika Cencen
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DOI: dx.doi.org/10.17504/protocols.io.ycgfstw
Protocol Citation: Veronika Cencen 2019. Glutaraldehyde fixation . protocols.io https://dx.doi.org/10.17504/protocols.io.ycgfstw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2019
Last Modified: February 22, 2019
Protocol Integer ID: 20584
Abstract
Fixation of adherent mammalian cells for AFM imaging
Guidelines
Prepare adherent cell culture on glass coverslips
Materials
MATERIALS
ReagentGlutaraldehyde, 25% solutionBio Basic Inc.Catalog #GC3870.SIZE.500ml
STEP MATERIALS
ReagentPBS

Protocol materials
ReagentGlutaraldehyde, 25% solutionBio Basic Inc.Catalog #GC3870.SIZE.500ml
ReagentPBS
ReagentPBS
Safety warnings
Glutaraldehyde requires a high degree of caution, even at low concentration! Carefully read SDS before use!

Classification according to Regulation (EC) No 1272/2008
Acute toxicity, Oral (Category 4), H302 Acute toxicity, Inhalation (Category 4), H332 Skin corrosion (Sub-category 1B), H314 Serious eye damage (Category 1), H318 Respiratory sensitisation (Category 1), H334 Skin sensitisation (Category 1), H317 Specific target organ toxicity - single exposure (Category 3), Respiratory system, H335 Short-term (acute) aquatic hazard (Category 1), H400 Long-term (chronic) aquatic hazard (Category 2), H411

Refer to SDS for details


Side effects include skin irritation.[4] If exposed to large amounts, nausea, headache, and shortness of breath may occur.[3] Protective equipment is recommended when used.[3] Glutaraldehyde is effective against a range of microorganisms including spores.[3][6]
As a strong sterilant, glutaraldehyde is toxic and a strong irritant.[15] There is no strong evidence of carcinogenic activity.[16] Some occupations that work with this chemical have an increased risk of some cancers.[16]
Before start
Ensure proper disposal container is available in the area
Perform entire procedure in ventilated fumehood, disinfect workspace and tools with ethanol
Use gloves and eye protection (fumehood should be sufficient to prevent inhalation so a mask is not required)

Rinse cells 3 times with appropriate buffer, such as PBS or Ringer's
ReagentPBS

Prepare Concentration2 Mass / % volume concentration glutaraldehyde in KMgH buffer (or other buffer used for your application). Pipette just enough of the dilution on top of the rinsed cells to cover the area. Let sit forDuration00:20:00
Glutaraldehyde chemical formula





Rinse at least 3 times with buffer, properly disposing of the diluted glutaraldehyde.
Expected result
Glutaraldehyde is appropriate for whole and unroofed cell fixation, so up to 100% should be fixed with decent crosslinking. For thicker samples use an addition of paraformaldehyde

Safety information