Feb 13, 2023

Public workspaceKarnovsky's Fixative

This protocol is a draft, published without a DOI.
  • 1University of Gothenburg
Jens Berndtsson
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Protocol Citation: Jens Berndtsson 2023. Karnovsky's Fixative. protocols.io https://protocols.io/view/karnovsky-39-s-fixative-ckiguubw
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: In development
We are still developing and optimizing this protocol
Created: December 15, 2022
Last Modified: February 13, 2023
Protocol Integer ID: 74024
Funders Acknowledgement:
National Microscopy Infrastructure
Grant ID: NMI (VR-RFI 2019-00217)
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Abstract
Karnovsky's fixative is a standard fixative devloped by M. J. Karnovsky and consist of: 2.5% glutaraldehyde, 2% formaldehyde, 0.02% sodium azide in 0.05 M Na-cacodylate buffer at pH 7.4.

FIXATION TIME:
  • 1 hour minimum, 2-3 hours preferred, can be left overnight at 4 °C.
  • For fixation of tissue, add to the fixative within 15 min of removal from patient.

References
Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy. Journal of Cell Biology, 27, 137-138A.
Guidelines
It is best to make 1L every time. Always two people – to check the measurements and so that one person can be closing tube lids while the other is pipetting to avoid the fixative evaporating.
Materials
ReagentAqueous Glutaraldehyde EM Grade 25% 100MLCatalog #16210
ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C

Protocol materials
ReagentAqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
In Materials, Safety warnings and 4 steps
ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
In Materials, Safety warnings and 4 steps
ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
In Materials, Safety warnings and 4 steps
ReagentMilli-Q water
In 4 steps
Safety warnings
Use caution, work in a fume hood, wear gloves, lab coat, and goggles.

ReagentAqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
H330 Fatal if inhaled.
H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.
H314 Causes severe skin burns and eye damage.
H318 Causes serious eye damage.
H302 Harmful if swallowed.
H317 May cause an allergic skin reaction.
H335 May cause respiratory irritation.

ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled
H315 Causes skin irritation.
H319 Causes serious eye irritation
H227 Combustible liquid.

ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
H300 + H310 + H330 Fatal if swallowed, in contact with skin or if inhaled.
H373 May cause damage to organs (Brain) through prolonged or repeated exposure if swallowed.
H410 Very toxic to aquatic life with long lasting effects.
Before start
Book a fume hood and label the aliquot tubes.
1000 ml
1000 ml
Mix the following in a 1000 ml beaker:
  • Amount100 mL of ReagentAqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • Amount125 mL of ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • Amount0.2 g of ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • Amount250 mL of Concentration200 millimolar (mM) Na-cacodylate buffer
  • Amount525 mL of ReagentMilli-Q water

Aliquote the mixture in:
  • 20 x Amount5 mL - 15 ml falcon tube labelled K5
  • 30 x Amount10 mL - 15 ml falcon tube labelled K10
  • 30 x Amount20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at Temperature-20 °C or short term (1-2 weeks) at Temperature4 °C

Clean up:
Rinse the used 1000 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
500 ml
500 ml
Mix the following in a 500 ml beaker:
  • Amount50 mL of ReagentAqueous Glutaraldehyde EM Grade 25% 100MLEMSCatalog #16210
  • Amount62.5 mL of ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • Amount0.1 g of ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • Amount125 mL of Concentration200 millimolar (mM) Na-cacodylate buffer
  • Amount262.5 mL of ReagentMilli-Q water

Aliquote the mixture in:
  • 10 x Amount5 mL - 15 ml falcon tube labelled K5
  • 15 x Amount10 mL - 15 ml falcon tube labelled K10
  • 15 x Amount20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at Temperature-20 °C or short term (1-2 weeks) at Temperature4 °C

Clean up:
Rinse the used 500 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
200 ml
200 ml
Mix the following in a 500 ml beaker:
  • Amount20 mL of ReagentAqueous Glutaraldehyde EM Grade 25% 100MLCatalog #16210
  • Amount25 mL of ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • Amount0.04 g of ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • Amount50 mL of Concentration200 millimolar (mM) Na-cacodylate buffer
  • Amount105 mL of ReagentMilli-Q water

Aliquote the mixture in:
  • 4 x Amount5 mL - 15 ml falcon tube labelled K5
  • 6 x Amount10 mL - 15 ml falcon tube labelled K10
  • 6 x Amount20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at Temperature-20 °C or short term (1-2 weeks) at Temperature4 °C

Clean up:
Rinse the used 500 ml beaker twice with 100 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.
100 ml
100 ml
Mix the following in a 100 ml beaker:
  • Amount10 mL of ReagentAqueous Glutaraldehyde EM Grade 25% 100MLCatalog #16210
  • Amount12.5 mL of ReagentPARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
  • Amount0.02 g of ReagentSodium azide (99.5%)BDH Chemicals Ltd Poole EnglandCatalog #9115010C
  • Amount25 mL of Concentration200 millimolar (mM) Na-cacodylate buffer
  • Amount52.5 mL of ReagentMilli-Q water

Aliquote the mixture in:
  • 2 x Amount5 mL - 15 ml falcon tube labelled K5
  • 3 x Amount10 mL - 15 ml falcon tube labelled K10
  • 3 x Amount20 mL - 50 ml falcon tube labelled K20

Dispose of the pipettes used for aliquoting in a hazardous waste bin.

Storage
Store long term (6-12 months) at Temperature-20 °C or short term (1-2 weeks) at Temperature4 °C

Clean up:
Rinse the used 100 ml beaker twice with 20 ml of water, disposing it in a liquid hazardous waste-jar for fixatives before putting the beaker in the dishwasher. Treat spillage as dangeorus and discard any contaminated saftey paper in a hazerdous waste bin. Spills outside of saftey papers should be cleaned up with paper and water which should then be discarded in a hazerdous waste bin.