Third time the charm?

This is my third trial on staining the Na+/K+-ATPase. The major problem I encountered in my first trial was from the sheath preventing the antibodies on penetrating and staining the ATPase. The major problem I encountered in my second trial was the poor tissue preparations from sectioning.

This time, after much research, I decided to use colleganse to dissolve the outer sheath enclosing the ganglia, and then stain the ATPase. However, the incubation length for colleganse could be tricky. Colleganse is a nonspecific enzyme that will digest will any collagen. This means initially the colleganse will digest the sheath surrounding the ganglia, but if you incubate your tissue in it for too long, the colleganse will then start digesting the cellular collagen and cause your tissue to lose its integrity. To determine the optimal colleganse incubation duration, I prepared three different samples each with a different incubation length, 6 hours, 12 hours, and 24 hours.

After much waiting, the results are finally in! It turns out that the 6-hour colleganse treatment was not sufficient enough to dissolve the outer sheath, but after the 24-hour treatment, the cell eventually loses its structure. As a result, the 12-hour treatment had the best compromise. Though, there is still dark area in the middle of the ganglion indicating and the antibody was still having trouble penetrating into the center...

This video shows the serial section images taken using the confocal microscope starting from the dorsal side of the ganglion.

With all the results in, I’m fairly happy with what I’m seeing, with the only thing I might want to change is trying to use an 18-hour colleganse treatment to see if that can improve my images. Now that I think I’ve got my staining protocol down, it’s time for some real results!