What is the minimal time needed? I alread read that people fix cryosection for 5 minutes or 10 minutes but I never saw any explanation for this times or experienced big differences myself. How fast is protein denaturation in sections? How much does it depend on the thickness of the section and the temperature of the fixative? I guess there is no over-fixation in precipitation fixation, but is there a under-fixation? How much time is necessary to remove all fat from the sections by acetone? Are there any additives to help fixation?
I agree with Liz, I have also stained without fixing with acetone and the staining came out nice. I do, however, fixed my sections for one hour in 4% PFA and washed in PBS prior to transferring to sucrose step. If you do not do this step, then you should fix with acetone after. 5 minutes should be ok. From my experience ice cold methanol is used mostly for cells but I might be wrong.
Hi Vivica, methanol or aceton not only fixes, but also permeabilizes the membrane. Thats why you should not fix longer than 5-10 mintues and also use less Triton-X or any other detergent, otherwise you might wash you proteins away...
The time plays role in the period just after your cryosectioning and before the fixation. The shorter this time= better results. Denaturation depends from protein itself, if not fixed immediately after the sectioning it will start denaturation process immediately (my experience). You will get the same results fixing it 5-10 min or longer. Focus on quicker fixation, soon after the cryosectioning. it will result with better IHC images.
I use ice cold methanol for 15 min in the -20, as I use 40um sections it helps to permebilize the tissue and I use this method with every FS I do. Also I use PBS with 1% Triton, I once ignored that step, but result was bad.
Thank you all for your anwers. Still the main question is open: Is there a reason (experimental data, literature) for fixing durations? For time between sectioning and drying I find different information (immediately as Sasha said vs. dry 3h or overnight before fixation). I think drying is also kind of a fixation since water gets lost during drying (depending on temperature and humidity in the room).
Every antigen is different and every antibody is different. The main questions are: where is the antigen located, how soluble is the antigen and, how tolerant is the antibody to denaturing of the antigen? Drying the tissue sections longer tends to help them stick to the slides better if you need to use a longer extraction time or higher concentration to Triton to expose the antigen, but it also tends to increase the background and make the structure look less pretty. So the only thing that I can suggest is that you start with a staining protocol for an antigen that is similar to the one that you want to visualize and then adjust it based on how well the structures is preserved and your signal to noise ratio for the staining.
As Karen has pointed out, there are several important considerations to make in optimizing your fixation protocol. My suggestion is to do a quick "dose response" experiment to standardize your method. I would do 3 or 4 different fixation times and compare them side by side after staining to see which works best. The important thing is to keep all other parameters the same and only change one variable at a time.
Fixation duration can influence how well your antibodies can permeate the tissue. It also can change the overall cytoarchitecture, especially if your tissue has a high lipid content. For example, spinal cord sections can look like swiss cheese if overfixed or dried for too long since the lipid content is so high.
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