Immunoperoxidase localization of albumin and fibrinogen in rat liver fixed by perfusion or immersion: effect of saponin on the intracellular penetration of labeled antibodies.
Immunoperoxidase localization of albumin and fibrinogen in rat liver was tested with perfusion or immersion fixation and saponin as a membrane permeabilizing agent. The distribution of albumin- or fibrinogen-containing hepatocytes was examined by light microscopy. Labeled antibody penetration was assessed by electron microscopy on transversely cut cryostat sections. Paraformaldehyde liver fixation by perfusion, followed by incubation of the sections with labeled antibodies together with saponin, demonstrated that albumin and fibrinogen were present in all hepatocytes; mainly in the Golgi apparatus and rarely in the endoplasmic reticulum, the ultrathin sections being labeled throughout their entire thickness. A constant labeling of the endoplasmic reticulum was obtained when saponin was added from the beginning of fixation. In the absence of saponin, albumin was seen in most of the hepatocytes but only at the periphery of the transverse sections, in a few Golgi apparatus, and in some parts of the endoplasmic reticulum; under this condition, fibrinogen was not visualized in the hepatocytes. Paraformaldehyde liver fixation by immersion showed the presence of albumin or fibrinogen in a few hepatocytes only, with irregular labeled antibody penetration. The use of saponin did not improve albumin and fibrinogen localization, except when the liver was poorly fixed. These results show that liver fixation by perfusion gives a homogeneous labeling of all the hepatocytes, whereas fixation by immersion leads to a heterogeneous labeling. Satisfactory results are obtained with saponin, which must be used to improve the penetration of labeled antibodies when the liver is fixed by perfusion. Saponin does not work when immersion is employed, at least under the conditions tested.
D000418:Albumins / Q000032:analysis*; D000818:Animals; D000906:Antibodies; D005340:Fibrinogen / Q000032:analysis*; D007124:Immunoenzyme Techniques; D008099:Liver / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins
D000418:Albumins; D000906:Antibodies; D012503:Saponins; D005340:Fibrinogen