Post-diaminobenzidine Treatments for Double Stainings: Extension of Sulfide-Silver-Gold Intensification for Light and Fluorescent Microscopy

Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3′-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions:

Diagnosis of Hirschsprung Disease

Diagnosis or exclusion of Hirschsprung disease (HSCR) is a frequent exercise in any pediatric hospital. Although HSCR may present at different ages and with varied clinical findings, the most common presentation is a neonate with severe constipation or signs of intestinal obstruction. A variety of diagnostic tests including contrast enema and anorectal manometry may be used as diagnostic screens, but diagnosis ultimately rests upon histopathological evaluation of a rectal biopsy. For the experienced pathologist, conventional hematoxylin-and-eosin-stained sections often suffice to exclude HSCR or establish the diagnosis. However, ancillary diagnostic tests such as acetylcholinesterase histochemistry or calretinin immunohistochemistry are complementary and extremely helpful in some cases. In this Perspectives article, we review the clinical and pathological features of HSCR, highlight those that are found in most patients, and discuss how to address particularly challenging aspects of the diagnostic workup.

Thalamocortical Afferents Innervate the Cortical Subplate much Earlier in Development in Primate than in Rodent

The current model, based on rodent data, proposes that thalamocortical afferents (TCA) innervate the subplate towards the end of cortical neurogenesis. This implies that the laminar identity of cortical neurons is specified by intrinsic instructions rather than information of thalamic origin. In order to determine whether this mechanism is conserved in the primates, we examined the growth of thalamocortical (TCA) and corticofugal afferents in early human and monkey fetal development. In the human, TCA, identified by secretagogin, calbindin, and ROBO1 immunoreactivity, were observed in the internal capsule of the ventral telencephalon as early as 7–7.5 PCW, crossing the pallial/subpallial boundary (PSB) by 8 PCW before the calretinin immunoreactive corticofugal fibers do. Furthermore, TCA were observed to be passing through the intermediate zone and innervating the presubplate of the dorsolateral cortex, and already by 10–12 PCW TCAs were occupying much of the cortex. Observations at equivalent stages in the marmoset confirmed that this pattern is conserved across primates. Therefore, our results demonstrate that in primates, TCAs innervate the cortical presubplate at earlier stages than previously demonstrated by acetylcholinesterase histochemistry, suggesting that pioneer thalamic afferents may contribute to early cortical circuitry that can participate in defining cortical neuron phenotypes.

Diagnostic Accuracy of Combined Acetylcholinesterase Histochemistry and Calretinin Immunohistochemistry of Rectal Biopsy Specimens in Hirschsprung’s Disease

Background. Acetylcholinesterase (AchE) histochemistry has been established as an accurate diagnostic tool for Hirschsprung’s disease (HD). In addition, calretinin immunohistochemistry is also reported as a reliable and adjunctive method to diagnose HD. We investigated the diagnostic value of combined AchE histochemistry and calretinin immunohistochemistry in rectal suction biopsies from HD and non-HD patients. Methods. We retrospectively reviewed 99 rectal suction biopsy specimens including 4 repeat biopsies from 95 patients (34 HD and 61 non-HD). Each specimen was evaluated with hematoxylin-eosin, AchE histochemistry, and calretinin immunohistochemistry. Results. Of 95 patients, only 21 (22.1%) showed some ganglion cells. All 61 non-HD cases revealed no abnormal AchE-positive fibers. Of 34 HD patients, 32 exhibited abnormal AchE fibers, but 2 showed no stained fibers. None of the tissues from the HD patients exhibited calretinin immunoreactivity. Test sensitivity and specificity of AchE histochemistry alone were 93.5% and 100.0%, respectively, while calretinin immunohistochemistry were 100.0% and 85.2%, respectively. Conclusions. AchE histochemistry is a good diagnostic method for HD, if feasible, and a combination of AchE histochemistry and calretinin immunohistochemistry will help increase the accuracy of the diagnosis of HD.

Experience of acetylcholinesterase histochemistry application in the diagnosis of chronic constipation in children

The aim of this study was to review our experience in applying acetylcholinesterase histochemistry for diagnosing colonic dysganglionoses in children. Patients and methods. We analyzed acetylcholinesterase histochemistry results of rectal biopsy specimens obtained from 85 children. The indications for biopsy were suspicion of Hirschsprung’s disease in neonates and infants (Group 1; n=21) and older children (Group 2; n=17); megarectum (Group 3; n=44); and colostomy (Group 4; n=3). Specimens were taken at 5 and 10 cm using endoscopic forceps or excised with scissors at 2.5 cm above the dentate line. Acetylcholinesterase activity was evaluated using Karnovsky-Roots method. Results. The diagnosis of Hirschsprung’s disease was confirmed in 17 children of the first group and in 3 of the second group. In the third group, 2 children were diagnosed with ultrashort-segment Hirschsprung’s disease and 3 children with intestinal neuronal dysplasia. In one case, acetylcholinesterase reaction was false positive. Hirschsprung’s disease was diagnosed in 2 children with colostomies; in one case acetylcholinesterase activity caused false-positive results. Colonic dysganglionoses were diagnosed in 78% of infants and in 14% of children over 1 year of age. The diagnostic specificity of acetylcholinesterase in Hirschsprung’s disease was 92%. Conclusions. 1) The analysis of acetylcholinesterase activity in children’s rectal biopsy specimens is a reliable method for diagnosing Hirschsprung’s disease, especially in infants; 2) This method of examination is irreplaceable in diagnosing ultrashort-segment Hirschsprung’s disease and remains the only method to confirm the diagnosis of this disease; 3) Acetylcholinesterase histochemistry is not sufficiently informative in diagnosing intestinal neuronal dysplasia type B, because authors applying other neurohistochemical investigation methods have reported higher incidence of this disease.