The authors have examined the sensitivity and detection efficiency of the three peroxidase methods that currently have the widest application in diagnostic immunohistochemistry: the peroxidase-antiperoxidase (PAP), the avidin-biotin complex (ABC), and the labeled avidin-biotin (LAB) methods. Sensitivity was evaluated by determining the highest useful dilution of polyclonal antiglucagon antibodies applied to formalin-fixed, paraffin-embedded human pancreas. Detection efficiency was evaluated by tabulation of the total number of positive (three or more positive cells) islets. On direct comparison, the LAB method exceeded the PAP and ABC methods in both sensitivity and detection efficiency, which were essentially equal. Titration of linking antiserum of the PAP method boosted its sensitivity and detection efficiency above that of ABC; the PAP had equal sensitivity to the LAB and exceeded it in detection efficiency. The authors conclude that comparisons of immunohistologic methods are meaningful only if both sensitivity and efficiency are considered along with the unique requirements of any single method.

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We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein liB (GLIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GLIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.The study of the cellular and tissue distribution patterns of DNA and RNA has been greatly facilitated by in situ hybridization. However, one limitation of in situ hybridization is its relatively high detection threshold, which is about 20 copies per cell. (1~ Recent work on the processing of RNAs has focused on the nuclear matrix, which appears to provide a specific and obligatory pathway for this processing from transcription to splicing up to and including release of translationally active message into the cytoplasm. (z-6) One observation supporting this hypothesis is the intimate association of the specific small nuclear (sn) RNAs and their associated proteins in spliceosomes, which are essential for splicing of pre-mRNA, with the nuclear matrix. (7,8) The inability to detect low copy numbers of RNA has hindered the study of the dynamics and subcellular organization of mRNA transcription, processing, and transport. Wang et al. (9) injected labeled 13-globin pre-mRNA into nuclei and observed discrete subnuclear compartmentalization of the signal. Others have noted a dramatic compartmentalization of pre-mRNAs in the nucleus. (1~ Detection of in situ PCR-amplified DNA from a single DNA molecule per cell has been described, (~'~1~ and the 100-fold amplification is readily detected by in situ hybridization analysis. The hot start modification to the PCR in situ technique was used to improve sensitivity by increasing the amount of specific target synthesis and reducing the nonspecific amplification that may follow extension of primers annealed to nontarget sequences (mispriming) and extension of primers after they anneal over parts of their sequence to form primer oligomers. This enhanced specificity allows the incorporation of a labeled nucleotide into the amplified product with the assurance that the resultant signal reflects tar...

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The most widely used non-enzymatic method for unmasking antigens prior to immunohistochen~ical staining is based on immersion of deparaffinized paraffin sections in citrate buffer and exposure to high temperature heating (100°C) with a microwave. In our experience, the benefits of this technique as applied to cytoplasmic proteins does not routinely translate to the nuclear estrogen and progesterone receptor (ER and PgR) proteins. The lifting or loss of portions of a breast biopsy section can seriously interfere with efficient scoring of invasive tumor cells. The use of low temperature restoration (80°C) in citrate buffer using a water bath avoids problems of tissue disruption and generally restores the immunoreactivity of ER and PgR proteins in a more uniform fashion than does the microwave approach. (The .I Histotechrzol 20: 155, 1997) based methods because it did not require special equipment and results could be obtained using established immunostaining methods within a few hours after receipt of the biopsy.The decreasing size of breast biopsies due primarily to increased use of screening mammography provided the impetus for development of techniques that obviated the need to dedicate a fresh frozen portion of the biopsy solely for the ER-ICA. Earlier reports indicated that enzyme digestion of routine paraffin sections of renal biopsies facilitated the immunohistochemical detection of immune complexes deposited in glomeruli (6). Subsequently, a similar "antigen retrieval" approach employing DNase and permitting use of H222 monoclonal antibodies on routine, formalin fixed, paraffin embedded sections was introduced (7). This provided an impetus for orher investigators to substitute pro-