Detecting proliferating cell nuclear antigen in archival rodent tissues.

The detection of proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, has lacked sensitivity in paraffin-embedded archival tissues fixed in formalin. An enhanced immunohistochemical procedure to detect PCNA has been successfully applied to rat and mouse tissues. Tissue sections are heated in a microwave oven in the presence of an antigen-retrieval solution of heavy-metal salts. Positive immunostaining of S-phase cells, an indication of DNA replicative activity, has been consistently obtained in tissues fixed for more than 24 months in formalin and in paraffin blocks stored for up to 19 months. Use of this technique will allow retrospective staining of rodent tissues from previously conducted toxicity and carcinogenicity studies.

The detection of proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, has lacked sensitivity in paraffin-embedded archival tissues fixed in formalin. An enhanced immunohistochemical procedure to detect PCNA has been successfully applied to rat and mouse tissues. Tissue sections are heated in a microwave oven in the presence of an antigen-retrieval solution of heavy-metal salts. Positive immunostaining of S-phase cells, an indication of DNA replicative activity, has been consistently obtained in tissues fixed for more than 24 months in fornalin and in paraffin blocks stored for up to 19 months. Use of this technique will allow retrospective staining of rodent tissues from previously conducted toxicity and carcinogenicity studies.
The role of sustained, enhanced cell proliferation in the induction of neoplasia has created renewed interest in and controversy over the mechanisms of carcinogenesis (1)(2)(3)(4). Lack of cell proliferation data and the cost of repeating rodent toxicity and carcinogenicity studies has precluded the resolution of this issue.
Identification of proliferating cells is indirectly assessed by labeling cells when they are in S phase of the cell cycle and is quantitated by the labeling index. Historically, agents such as tritiated thymidine and bromodeoxyuridine, a thymidine analog, were administered before sacrifice and incorporated into cells during DNA synthesis (S phase of the cell cycle). More recently, proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase-6 (5-7), has been shown to be a highly stable protein found in all mammalian tissues (8) and serves as an endogenous cell replication marker. PCNA is present in normal and transformed proliferating tissue culture cells, cryostat sections, and alcohol-and formalin-fixed paraffinembedded tissues (8)(9)(10)(11)(12)(13) tics in rat liver fixed overnight in formalin allow the identification of cells in the quiescent (GO), interphase (G1, S, G2) and mitotic stages of the cell cycle (14). However, detection of PCNA has lacked sensitivity in paraffin-embedded tissues fixed in formalin for 1 week or more, as commonly occurs in toxicity and carcinogenicity bioassays.
An enhanced immunohistochemical procedure to detect PCNA has been successfully applied to formalin-fixed, paraffin-embedded archival rat and mouse tissues (15). Tissue fixation in formalin varied from 1 week to 27 months. The unmasking of antigenic sites was accomplished by heating tissue sections in a microwave oven in the presence of a commercially available antigen retrieval solution of heavy metal salts before PCNA immunostaining.
Current modifications to the PCNA immunohistochemistry to improve background staining problems included changes in the protein-blocking reagent, from 1% normal goat serum to 0.5% nonfat powdered milk, and the replacement of the link antibody to a biotinylated anti-mouse IgM (,u-chain specific; Vector Laboratories, Burlingame, CA). Microwave times vary depending on tissues and chemical treatment. Two to five minutes on the highest power (700 watts) was found to be sufficient for all tissues tested.
Intense nuclear staining of S-phase cells, an indication of DNA replicative activity, was consistently obtained in tissues fixed for 27 months in formalin and in paraffin blocks stored for up to 19 months ( Figure  1B, D). This technique has been successfully applied to liver, kidney, spleen, forestomach, glandular stomach, small intestine, esophagus, testis, and lung. Late G1 cells showed light nuclear staining of PCNA, whereas cells in G2 and mitosis had diffuse cytoplasmic staining. Archival tissues stained in the absence of the antigenretrieval solution showed minimal to no nuclear staining ( Figure 1A, C). Similarly, predigestion of tissue sections with a battery of proteolytic enzymes did not unmask antigenic sites in the absence of pretreatment with the antigen-retrieval solution. Formalin fixation results in protein cross-links in tissues. Continued formalin exposure increases the degree of cross-linking and masks antigenic sites such that they cannot react with specific antibodies. The antigen-retrieval technique based on microwave heat-ing of tissue sections in the presence of heavy-metal salts permits enhancement of immunohistochemical staining of tissue antigens in a variety of formalinfixed, paraffin-embedded tissues (16). Use of this technique will allow retrospective staining of rodent tissues in previously conducted toxicity and carcinogenicity studies in which the animals were not pretreated with a labeling agent.