Help with Histokinet for mouse brain - (Feb/28/2007 )
Hi, I'm hoping someone could offer some help on histokinet steps for mouse brain. It appears to be embedded in the wax properly but when I section the tissue looks all wrinkled. It doesn't seem to be problem with the microtome as the wax slices fine. Did I overfix? Is it not fully infused with wax?
The histonkinet steps are:
1) fixative 5 mins
2) 1/2 hour each for 50%, 70%, 95% EtOH
3) 3x 1hour 100% EtOH
4) 1:1 EtOH histoclear for 30 minutes
5) Histoclear 1.5 hours, then 2 hours
6) Paraffin 2.5 hours, then 4 hours
Does temperature have a huge effect? We are using low temp melting wax (54-57C) but the machine is set at 62C. Is this extra heat damaging? Unnecessary?
I've done cryosectioning before and worked with ready-made paraffin sections so I'm pretty sure I understand what's going on in each step, just having trouble working out the timing.
What is your fixative? 5 minutes is a very short fixation time. However, I think the main cause of your problem may be that you are dehydrating your samples for too long. I've never worked with brain, but I had similar problems with mouse ovaries. I decreased the dehydration time and got a huge improvement in the quality of the sections.
In my experience, high temperature does not cause problems with sectioning. I use paraplast and heat it to ~65, which is higher than recommended.
When I'm just sectioning purely for histology, so I need perfect sections, but NOT for IHC, I use this protocol for mouse ovaries. Maybe it will work for brain too.
Carnoy's Fluid 30-60 minutes (I use 30, but ovaries are much smaller than brain)
(Carnoy's fluid is EtOH 60 ml, chloroform 30 ml, and acetic acid 10 ml).
100% EtOH 3 x 30 minutes
Toluene 15-30 minutes
Paraplast 1 hour (or longer is fine)
Sorry - my fault on the fixation time - I fix for 48 hours in 4% PFA at 4 degrees.
Thanks, I will try shorter dehydration steps, I had a feeling that had something to do with it but the whole temperature thing is throwing me off - like I was "frying" the brains or something.
Can I ask, what is the purpose of the chloroform? Does it act as a fixative? We have it in the lab, but its never used as we use halothane or isoflurane as inhalation anesthetics - I have been wondering if it would serve any purpose or if we should get rid of it and avoid having it around.
The chloroform added to EtOH and acetic acid helps to decrease the amount of cell shrinkage you would see if you try to fix with EtOH alone.
For your fixation step, you might try decreaing the time on that as well. I fix ovaries in either Neutral buffered formalin or Bouin's, and I leave them for only 1 hr.