I am doing immunohistochemistry (IHC) in Prefrontal cortex of brain section slices after fixing by formalin 4%. My IHC images have so many white vacuole, has anyone who has experienced this problem?
Are you using free floating sections?Where are the white vacuoles in the tissue?You may want to review your fixation protocol and also check how you are slicing the brain, artifacts usually arise when the tissue is sectioned with a blunt blade. If you are using frozen sections or cryoprotection with sucrose make sure the tissue completely sits at the bottom of the tube/container after cryopreservation otherwise the tissue won't cut so well. I am not sure what exactly would be a great response, perhaps if you add more information by responding to the questions I have raised
It is likely a fixation problem, I suggest using the pH shift method for fixation. Method is described in my chapter titled, "Specimen Preservation for Confocal Light Microscopy." in the Handbook of Confocal Microscopy
Judging by the text of your question, you appear to have fixed the brain tissue specimens in 4% formalin, as oppose to cryopreserving. Therefore, following factors may have influenced the quality of your specimens.
(1)Was the formalin made in a buffer with an appropriate pH. (2) The age of the formalin solution is known to influence, particularly a sensitive tissue such as brain. (3)Did you process the post-fixed tissue specimens in a vacuum filter processor. If so, processing tissue specimens at over 59 degree Celsius is known to have deleterious affects on the integrity of specimens. (4) I also agree with Denver that a blunt microtome blade could have distorting affects.
I agree with suggestions of Dr Ashraf Iman, Especially, No 3 would be most important one you should try. If they are air vacuoles in tissues, they can be removed by a vacuum filter processor. Good luck
Thank you very much for your informations, I am using formalin 4% for post fixing 5 days in 4 Celsius after perfusion with saline 0. 9% for free floating sections staining.