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• 1

  Erdrin Azemi

  07 July 2009 | 18:46

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  I work with rats but I believe you can use the same protocol for mouse.

  -Perfuse the animal with cold (4 degrees) 4% Paraformaldehyde transcardially (we use ~300ml cold 1xPBS and ~300ml cold 4% Paraformaldehyde – you might want to use half of the amounts for mice – using a peristaltic perfusion or gravity feed pump connected with a 18 gage needle through the left ventricle and by clamping the descending aorta)
  -After perfusion decapitate the animal and remove the brain carefully using bone roungers to break off the skull
  -Post fix the brain into a vial with 4% Paraformaldehyde for 24hrs (in 4 degrees)
  -After washing in PBS immerse the brain into a vial with 30% Sucrose until it sinks in the bottom of the vial (in 4 degrees) – about 2-3 days
  -Use O.C.T for cryoprotection if using a cryostat or leave the brain as is for microtome floating sections
  -For IHC block for 45 min and then leave the tissue sections overnight in primary antibody so it penetrates through the tissue followed by 1 hour in secondary antibody and by 15 min in nuclear dye solution.

  I hope this helps! – I follow this protocol for rat brain tissues and it has always worked.
  Let me know if you need more info or if you have any questions about the protocol.

  Good luck!