Development of T and B cell areas in peripheral lymphoid organs of the rat.

In the present study the early development of peripheral lymphoid organs (spleen, popliteal lymph node, mesenteric lymph node and Peyer's patches) is described in terms of homing patterns of T and B cells, demonstrated with immunohistoperoxidatic detection of characteristic membrane antigen in normal rats and with routine histology in neonatally thymectomized rats. In the first days after birth the peripheral lymphoid organs are almost exclusively populated by T cells. After neonatal thymectomy lymphocytes appear in the dome areas of Peyer's patches from four to six days after birth, in mesenteric and popliteal lymph nodes lymphocytes are found in the outer cortex from day 6 and day 8 respectively and in the marginal zone of the spleen from eight days onwards. These lymphocytes showed no membrane staining when reacted for T antigen with immunohistoperoxidatic techniques. The morphological evidence for considering Peyer's patches of rats as central inductive sites for the generation of B cells is poor. The discrepancy in the order of appearance of T and B cell (sub)populations in spleen compartments in normal ontogenetic development and lethally irradiated, stem cell reconstituted animals is discussed.

Periodate or glutaraldehyde for preparing peroxidase conjugates?

Conjugates of horseradish peroxidase with antibodies (anti-human IgG (H + L)) or their Fab' fragments were prepared according to the newest modification of the periodate (P-) method or the two-step glutaraldehyde (G-) method. The conjugates were analysed by gel chromatography and subsequently tested in three different applications. For tissue immunohistochemistry on sections of lupus erythematosus skin, G-conjugates were preferred to polymeric P-conjugates. In ELISA for detection of human antibodies against penicillin P-conjugates were superior to G-conjugates. For the detection of surface Ig on lymphoid cells both types of conjugate were more or less equally suitable. A scheme for the most suitable combinations of method of preparation and field of application is given.

Peroxidase-conjugate chromatography isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel.

Fractionation abilities of polyacrylamide-agarose gel (Ultrogel) and dextran gel (Sephadex) column chromatography were compared in isolating horseradish peroxidase conjugates, prepared using two different methods. Utrogel AcA-44 provides an efficient separation of monomer conjugated and nonconjugated immunoglobulins resulting from the two-step glutaraldehyde procedure, Sephadex G-200 does not. Both types of columns eluted the polymer conjugates resulting from the periodate procedure in the void volume; these were hardly isolated from the small amount of monomer conjugate. Unreacted horseradish peroxidase, present in very low quantities after the efficient periodate method and in large amounts after the glutaraldehyde procedure, was separated by both gel types.

Autoradiography and immunohistoperoxidase techniques applied to the same tissue section.

A method is described which allows simultaneous detection of two different compounds in tissue sections by combining autoradiography and immunohistoperoxidase techniques.

A new method for the cytochemical demonstration of peroxidase for light, fluorescence and electron microscopy.

A new technique for the cytochemical demonstration of peroxidase is presented. Monomeric homovanillic acid is converted by H2O2 and peroxidase into its dimeric form, which is then precipitated as a complex salt of lead and rhodamine 6G or rhodamine B. The reaction product can be visualized by conversion to lead sulphide or viewed directly under the fluorescence microscope, since it emits a red fluorescence when excited with green light. The reaction is rapid and results in good localization at the cytologic level of peroxidase activity in granulocytes. The technique can be applied for the ultrastructural localization of enzymatic activity, but in its present form it does not match the localization sharpness of the diaminobenzidine method. This fluorescent cytochemical technique will also detect horseradish peroxidase activity and may provide a usefull probe in peroxidase immunohistochemistry. The principle of complexing metal salts with fluorescent dyes may find a more general application in enzyme cytochemistry.

Antigen-coupled beads adherent to slides: a simplified method for immunological studies.

Small amounts of antigen-coupled beads adherent to object slides provide a simple, quick, economical and sensitive immunohistochemical means of detecting antibodies in serum by both immunofluorescence and immunohistoperoxidase procedures. Sensitivity increases with decreasing quantities of antigen-coupled beads, as was demonstrated in the fluorescence procedure.

Some aspects of immunohistoperoxidase reactions in the agarose bead model system.

Agarose beads that serve as matrices for antigens can be useful tools, not only for studying the quantitative aspects of immunohistoenzyme reactions but also for detecting antibodies. The present study focuses on localization of the ultimate reaction product within the beads; this factor is important when quantitative aspects of immunohistoenzyme reactions are under investigation. Localization varies with respect to the concentration of CNBr used to activate the beads. In addition, a method is described that facilitates the use of indirect immunohistochemical bead procedures and reduces the amount of antigen (-coupled beads) needed. These advantages may be particularly helpful when these methods are used for serologic detection of specific antibodies on a large scale.

Serodiagnostic application of immunohistoperoxidase reactions on antigen-coupled agarose beads.

Agarose beads to which antigens were covalently bound were subjected to indirect immunohistoperoxidase procedures. For detection and titration of serum antibodies against bovine gamma globulin (BGG) this method appeared to be specific and sensitive. One advantage is that no special instruments are needed. As an example of diagnostic applicability the system was successfully used for demonstration of antibodies in human serum containing antibodies against the trematode Schistosoma mansoni. At the microscopical level the technique is suitable for study of basic problems. Microspectrophotometric absorbance scanning of the beads revealed that the method can provide quantitative information, and is probably capable of quantifying stoichiometric relations in immunological reactions.

Agarose beads as matrices for proteins in cytophotometric investigations of immunohistoperoxidase procedures;.

Quantitative aspects of direct immunohistoperoxidase procedures were studied in a model system consisting of agarose beads to which antigens or antibodies had been coupled. It could be proven that the final amount of reaction product resulting from the histoperoxidase reaction with 3,3-diaminobenzidine-tetra HCl in a bead was linearly related to the volume of the beads and to the staining time. This implies that protein-coupled agarose beads are a suitable model for the study of stoichiometric aspects of immunologic reactions in immunohistochemistry as well as in general immunologic methods when peroxidase is used as the protein marker.