In our lab we use Ottix Plus and Ottix shaper dehydration and deparrafination media, as more safe substitutes of Ethanol and Xylene.
However, when I was fixing and staining some muscle samples, I saw the morphology was quite bad. Can this be because of ottix, or is my fixation (10% formalin solution for 48h) the cause?
First of all, make sure the Formalin not concentrated, frequent uses of the Formalin sometimes becomes turbid due to production of Paraformaldehyde and this decreases the strength of the solution. To avoid this issue just filter it before use.
Second, wash the tissues in running water to remove all traces of fixative from tissue to be embedded.
For me I'm always using traditional method and I had great results with high quality, Dehydration starting from 50% for soft tissues like spleen or liver and 70% for the others such as heart or bone. The important thing in this stage is to remove all water from the tissue, So normally when I used the series of Ethanol 70%,80%,90% and 100% I add 10mm layer of Anhydrous copper Sulphate in the bottom of the last jar (100%) as an indicator of water so this reagent turn blue colour when water is present, in this way you will know the tissue has water and you need to repeat the last step of dehydration 100% two times to make sure all water gone.
Regarding to Clearing step I think there is no agent effective like Xylene, the Xylene is cheap when you compared with Chloroform!, so I would recommend to use the tradition method, put the tissues after Dehydration in Xylene for 1hr two changes to get very clear and transparent tissues.
I hope this was helpful and if you have any question don't hesitate to ask.
Adil
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