I was wondering if you can help me. I am growing some primary neurons and I have read many different protocols suggesting various fixatives.
Does anyone know why you would chose to add sucrose to a 4% formaldehyde fixative solution?
I want to try and maintain as much of the neuronal morphology as possible, are there any recommendations?
Thanks
-Weezie - newbie of the day

The addition of sucrose is often used to maintain a better tissue morphology. Although I've only seen this done with tissue samples, it could be worth trying on your cells. Just do an internet search and you can find groups that have done this and they explain why. As for your fixative, different methods maintain different aspects of cellular morphology. You need to determine which fix is best for what you are trying to image. Also keep in mind that certain antibodies are very particular and won't work with certain fixes. You may find yourself having to compare different methods...but at least this way you'll get the best!

we have found that using acetone for permeabilising the neurons preserved the morphology especially the processes which looked nice.

However, you do have to try out the different fixatives and permeabilising solutions to figure the one that suits your neurons.

Great...thanks for the help.

I don't work with neurons but for my protocol I had to fix lymphocytes. I was reading everything I could find those days to make my protocol. I had come across one article about fixation of neurons. See if it helps; but I think, you will have to tailor your technique trying any of the method that sounds good enough for a try.

Calibration and standardization of microwave ovens for fixation of brain and peripheral nerve tissue.
Login GR, Leonard JB, Dvorak AM.

Department of Pathology, Harvard School of Dental Medicine, Boston, Massachusetts, 02215, USA. glogin@bidmc.harvard.edu

Rapid and reproducible fixation of brain and peripheral nerve tissue for light and electron microscopy studies can be done in a microwave oven. In this review we report a standardized nomenclature for diverse fixation techniques that use microwave heating: (1) microwave stabilization, (2) fast and ultrafast primary microwave-chemical fixation, (3) microwave irradiation followed by chemical fixation, (4) primary chemical fixation followed by microwave irradiation, and (5) microwave fixation used in various combinations with freeze fixation. All of these methods are well suited to fix brain tissue for light microscopy. Fast primary microwave-chemical fixation is best for immunoelectron microscopy studies. We also review how the physical characteristics of the microwave frequency and the dimensions of microwave oven cavities can compromise microwave fixation results. A microwave oven can be calibrated for fixation when the following parameters are standardized: irradiation time; water load volume, initial temperature, and placement within the oven; fixative composition, volume, and initial temperature; and specimen container shape and placement within the oven. Using two recently developed calibration tools, the neon bulb array and the agar-saline-Giemsa tissue phantom, we report a simple calibration protocol that identifies regions within a microwave oven for uniform microwave fixation. Copyright 1998 Academic Press.

PMID: 9654457 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/sites/entrez?D...Pubmed_RVDocSum

what did you do today honey?
Oh i microwaved someones brain.
thats nice dear.

sometimes i forget just how bizarre what we do really is

dom

acetone, acetone/methanol, paraformaldehyde - i always start with those three and work out from there (never heard the thing about sugar before)

The sucrose is needed only when you are going to freeze the tissue later. Is a cryoprotectant, useful when you're sectioning with a cryostat or something of the kind. Hope this helps

The sucrose is needed only when you are going to freeze the tissue later. Is a cryoprotectant, useful when you're sectioning with a cryostat or something of the kind. Hope this helps