Fig. 7.1
(a) Pimonidazole binds to general proteins under ischemia due to adduction, and the antibody reacts to the protein adduct s of reductively activated pimonidazole (PARaPi). (b) Experimental protocol shown in this chapter; one hour after the intraperitoneal injection of pimonidazole, in vivo cryotechnique (IVCT) was performed at 30 s of hypoxia . (c) Schematic representation of the hepatic lobul e after 30 s of hypoxia, which is immunostained for PARaPi in pericentral zone . (d–g) HE staining images (d, e) and immunostaining images for PARaPi (f, g) in mouse liver tissues after 30 s of hypoxia. The PARaPi immunostaining is obtained around central vein s (CV; arrowheads in g), but it is very weak around interlobular veins (ILV). Precise data have been reported in the previous paper (Terada et al. [14]). Bars: d–g, 50 μm
7.2 How to Detect PARaPi Immunohistochemically in IVCT Tissues?
We demonstrate brief protocol of immunohistochemical approach for the IVCT tissue samples. Pimonidazole was intraperitoneally injected into mice. One hour after the injection of pimonidazole , the mice were anesthetized with pentobarbital. Their respiration stopped by inhalation of nitrogen gas in plastic bags, and at various interval times after their respiration completely stopped, their livers were surgically exposed, and an isopentane –propane mixture precooled in liquid nitrogen was directly poured on them, as described in the procedure for IVCT [1, 4].
The frozen liver tissues were freeze-substituted in acetone containing paraformaldehyde and embedded in paraffin . For immunostaining of PARaPi, deparaffinized sections were immunostained with commercially available FITC-conjugated anti-PARaPi monoclonal antibody followed by horseradish peroxidase (HRP) -conjugated anti-FITC monoclonal antibody. The immunoreaction products were visualized using the peroxidase enzyme reaction with metal-enhanced 3,3′-diaminobenzidine (DAB). This immunoperoxidase staining enables us to obtain better sensitivity.
7.3 Immunostaining for PARaPi in Normal and Hypoxic Liver Tissues
In normal liver tissues with IVCT, cells immunopositive for PARaPi were rarely observed, indicating oxygen tension in liver tissues was well retained under normal blood circulation .
At 30 s of hypoxia (Fig. 7.1b), erythrocytes formed typical rouleaux in sinusoids (Fig. 7.1d, e), and the PARaPi was strongly immunostained than in the normal liver , especially in some hepatocytes of the pericentral zone (Fig. 7.1f, g). The further from the central vein , the less immunoreactivity of PARaPi was detected, indicating that a change of the hypoxic state was detectable within 30 s by IVCT, as schematically demonstrated in Fig. 7.1c.
After 1 min of hypoxia , most hepatocytes in all hepatic zone s were immunopositive for PARaPi, indicating that they were in a hypoxic state. The relative intensity of PARaPi immunoreactivity was stronger in hepatocytes of the pericentral zone than in those of other zones, and hepatocytes showed various degrees of immunoreactivity against PARaPi even in the periportal zone. To evaluate whether such changes of cell membrane permeability occurred in such hypoxic cell s , serial sections were possible to be immunostained for mouse serum IgG1 . IgG1 was immunostained in blood vessel s and intercellular spaces, but not inside the hepatocytes, indicating that the changes of PARaPi immunostaining were not due to diffusion of cytoplasmic proteins into extracellular space s .
7.4 Application of IVCT Followed by FS for Immunoblotting Analyses
In addition, immunoblotting analyses can be done on FS samples. By immunoblotting, the intensity of immunoreactivity for PARaPi was observed after hypoxia for 3 min, whereas it was much weaker with the normal liver tissues in vivo. Thus, it was possible to examine the same tissue samples by immunohistochemistry and immunoblotting analyses.
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