That is a pretty good reference. Most protocols are found within the methods sections of histology papers.

Fixation time will depend on the type of fixative as some penetrate faster than others. I use 10% neutral buffered formalin (10% NBF) the most often for basic histology, but another type of fixative might be better for whatever application your are doing. For 10% NBF - For adults, immediately after euthanasia, I make an incision in the ventral body wall, cutting only through skin and muscle, from the anus to near the pectoral girdle. Then I use a small pipette to flush fixative through this incision into the body cavity. I also use the pipette to flush fixative into the mouth. These flushing steps help to get the fixative near deep tissues that would otherwise be difficult for the fixative to reach. That "immediately after euthanasia" is important; you want to do these steps and get fixative introduced quickly to prevent or limit autolysis. Then immerse the fish in at least 10X volume of fixative solution. I then put the container on a slow moving orbital shaker for ~24 hrs. This agitation also helps the fixative to penetrate the tissues. You can then store it in fixative until you process it. For formalin, it is stable as long as the pH remains neutral.

When you are ready to process the specimen(s), you will need to decalcify with either 10% formic acid or EDTA if fish have a significant amount of bone development; generally after 30dpf. Look up papers from Keith Cheng out of Pennsylvania State University for some good methods.

The processing schedule in the link you posted should do well. I tend to do 1 hr for each ethanol step, and I do 3 100% ethanol steps to make sure all of the water is out of the tissues. Similarly, for the clearing steps, I'll do either 3 steps of xylene (45 min each) or ClearRite3 (1.5 hrs each). Again, to make sure all of the ethanol is out of the tissue so that paraffin can get in. All of my paraffin steps are 45 min to 1 hr under vacuum. Paraplast is a type of paraffin wax that has some additional additives to help it penetrate tissue faster. I like it and use it but you can use whatever you have or prefer.

Our lab's staining protocol has the same basic elements, but it is quite a bit different than the one on this website so I can't comment on how well that particular protocol works.

Hope that helps!

- Melissa