Karnovsky’s Modified Fixative
Mark Ellisman, Mason Mackey, Marianne Martone
Published: 2023-07-14 DOI: 10.17504/protocols.io.261genwoog47/v1
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Abstract
Fixative for mice and rat tissues to be used during perfusion and immersion fixation.
Before start
Make sure heating plate for dissolving paraformaldehyde is between 60-70° C.
Steps
1.
Add 2.0 grams paraformaldehyde to a 100mL Erlenmeyer flask.
2.
Add 35mL of DDH2O to 100mL to a Erlenmeyer flask.
3.
Heat paraformaldehyde in DDH2O to 60-70ºC. Use stir bar to mix.
4.
Add 2-3 drops 1N NaOH.
5.
Filter solution with #1 Whatman filter into a 100mL graduated cylinder.
6.
Add 10.0 ml of 25% glutaraldehyde.
7.
Dilute with DDH2O to a total volume 49.0 ml.
8.
Add 1.0 ml of 0.204 M CaCl2-2H2O.
9.
Add 50 ml 0.3M cacodylate buffer stock solution at pH 7.4.
10.
Volume should be 100 ml. Transfer fixative into a 100mL Erlenmeyer flask.
11.
Keep in bath water 35ºC.
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