Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

Abstract

The quick-freezing method often used for physical fixation also contributed enormously to the advancement of morphology, but it could not provide enough information about the dynamic morphological changes in vivo in living animal organs. Therefore, we developed the in vivo cryotechnique in 1995, which could directly cryofix organs in vivo under anesthetized conditions without stopping blood circulation or producing effects of anoxia. We have already reported new findings about the in vivo ultrastructures of living animal organs with the cryotechnique followed by freeze-substitution or replica preparation. Recently, it has also been applied to other morphological analyses in vivo, including immunohistochemistry and FISH, for obtaining dynamically functioning structures of cells and tissues at a light microscopic level. In such experimental processes, the in vivo cryotechnique has been shown to have the added benefit of direct antigen-retrieval effects since it reduces several steps of retrieval treatments which are required in common paraffin-embedded samples prepared by the conventional fixation and dehydration. In conclusion, the in vivo cryotechnique allows us to investigate the functioning real morphology of living animals, which was technically difficult to be observed before, and perform dynamic immunohistochemistry of cells and tissues in various animal organs in vivo at ultimate time-resolution.

Extracellular space in mouse cerebellar cortex revealed by in vivo cryotechnique

“…The fixed cerebellar tissues were dehydrated in a graded series of ethanol and embedded in Quetol 812 (Nisshin EM, Tokyo, Japan), as described before . Other cerebellar tissues of six anesthetized mice used for FQF were surgically removed, immediately cut into small pieces with razor blades, and quickly frozen Ϸ5 minutes after the tissue resection by plunging them into the isopentane-propane cryogen (Ϫ193°C), as described previously (Ohno et al, 2004b).…”

“…fixed in vivo by using an isopentane-propane cryogen in combination with a knife precooled in liquid nitrogen, called a "cryoknife" (Ohno et al, , 2004b. The method has been applied to various organs of living mice to reveal their functional morphology under physiological or pathological conditions (Ohno et al, 2004a;Terada et al, 2006a).…”

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

“…The specimens were prepared as described previously (Ohno et al 2004c(Ohno et al , 2005a. BrieXy, the left lobe of each liver was removed from four starved mice under anesthesia and cut into small pieces.…”

“…BrieXy, the left lobe of each liver was removed from four starved mice under anesthesia and cut into small pieces. The pieces were quickly frozen by plunging into isopentane-propane cryogen (¡193°C) immediately (FQF-1 min) or 5 min (FQF-5 min) after the tissue-resection, as reported before (Ohno et al 2004c). After following freeze-substitution in acetone containing 2% PFA, they were embedded in paraYn wax as described before (Ohno et al 2005a).…”

“…The livers of four mice were cryoWxed by IVCT immediately after the liver exposure, as described previously (normal blood circulation; IVCT-N) (Ohno et al 2004c). For the experimental ischemia group, before IVCT, portal veins and hepatic arteries of four other mice were ligated at the porta hepatis for 80 min (ischemic condition; IVCT-IC).…”