A method is described for the fixation of blocks of tissue for use in studies employing immunofluorescence. This method consists of fixing thin blocks in 95% ethanol and carrying out the subsequent dehydration and clearing at refrigerator temperatures (4°C). Thereafter, embedding in paraffin and sectioning by the standard microtomy is easy. This method results in preparations which are histologically more precise in the localization of antigen or antibody than preparations of frozen tissues; and with rabbit antibody and bovine serum albumin, the sensitivity of detection is enhanced. Bovine serum albumin can be found for longer periods after injection than is possible with frozen sections. Other antigens for which this procedure has proved satisfactory are bovine gamma globulin, horse ferritin, influenza A virus, diphtheria and tetanus toxoids. Hen's ovalbumin deteriorated. New antigens or new antibodies should be tested before being committed to this method.

The immunogenic content of the afferent lymph stimulates structures in the lymph node. Thus, a better knowledge of the processes of lymph flow and filtration in the organ should help us better understand various aspects of the node's function. To gain this understanding, we analyzed the distribution of flow in rat node draining areas locally injected with a small dose of China ink. Because the lymph-flow pattern is likely related to the overall architecture of the node, we simultaneously studied its morphology. Indeed, while the different structures of the node are known, some aspects of its overall architecture need to be resolved. The present work aimed to accomplish this by an analysis of semiserial sections of nodes from various anatomical locations in normal rats; the sections were stained by the Dominici technique or silver-impregnated.With respect to their architecture, the nodes could be distinguished into those with either a discontinuous or a continuous subcapsular sinus and peripheral cortex. These are referred to here, respectively, as segmented and nonsegmented nodes. In the segmented nodes, the subcapsular sinus with the peripheral cortex is separated by "gaps," in which medullary sinuses reached the capsule. Further, a node appears to be divided into one or more "physiological compartments," each one representing a nodal area related to an opening of an afferent lymphatic.The findings on China-ink distribution indicate that the lymph-flow pattern varies in different nodes and is determined by the particular architecture of a node, i.e., the lymph flow in a given node aligns itself along the pattern of segmentation of the organ. The findings suggest that the lymph content is first held by the endothelium lining the inner wall of a restricted area of the subcapsular sinus in a concentration which decreases with the distance from the related afferent lymphatic opening. Part of the content, possibly its nonimmunogenic fraction, would later be released to flow further along the sinus. It would then be phagocytosed by the macrophage accumulation located in the portions of medullary sinuses into which the lymph enters from the subcapsular sinus. The lymph thus filtered then flows along the medullary sinuses and leaves the organ. The latter findings also indicate that a node is divided into physiological compartments, each one being stimulated by the lymph from a given afferent lymphatic opening. As the immunogenic content of the lymph can differ from one lymphatic to another, this explains the frequent variations in similar structures located in different areas of a given node. Hence, the pattern of distribution of the openings of the afferent lymphatics of a node can account for the particularities of its overall architecture and its division into physiological compartments.

A light-microscopy study of mated female snow crabs (Chionoecetes opilio) was conducted to investigate the site of fertilization and to resolve how multiple ejaculates are stored in the spermathecae. In its basic configuration, an ejaculate consisted of a layer or patch of spermatophores enclosing mature spermatids that was capped by a relatively large volume of amorphous matter, which in turn could include a patch of spermatophores containing immature spermatids. Up to 10-12 ejaculates were stored in the spermathecae with the largest loads. An ejaculate was initially deposited in the intermediate chamber and ventral part of the spermatheca, and was displaced toward the dorsal part of the spermatheca when a new ejaculate was inserted. Ejaculates were neatly stratified along the ventrodorsal axis of spermathecae with small to moderate loads, but they were disrupted and the storage pattern was disorderly in spermathecae with large loads. Ejaculate stratification favors last-male sperm precedence and single-male paternity. However, multiple-male paternity might occur in females with large spermathecal loads, in part because several ejaculates can co-occur close to the oviduct opening. Mixing of male and female gametes in preparation for oviposition, and probably also fertilization, occurs to some degree in the ovaries.

Scenarios have been proposed to explain how lymphoid components of a lymph node favor the encounter of a drained antigen with a circulating competent naïve lymphocyte to trigger a primary immune response. However, these scenarios rest on incorrect concepts about the organ. This situation resulted from a loss of interest for studies on in vivo lymphoid organs due to a widespread switch, decades ago, to work on suspended lymphoid cells. However, an in vivo holistic study of the organ continued in our laboratory. The present review synthesizes resulting knowledge on lymph node morphology and global functioning. We show that the opening of an afferent lymphatic vessel into the subcapsular sinus is the focal point from which the related portion of a lymph node-a node compartment-is developed. As to the formation of a compartment's lymphoid components, it is neonatally orchestrated by the dichotomic nature and distribution of antigens in this subcapsular sinus, which determines a dichotomic recruitment of circulating cells and the compartment's architectural complexity. The transport process of an antigen from a given tissue territory into restricted sites of the draining compartment further defines its local morphological features and activities, while providing the possibility to reduce the wandering of a short-lived naïve cell through innumerable target-devoid sites. We also explain that the nodal lymphoid components are not implicated in the triggering of primary responses, but are rather products of such responses. Scenarios for the triggering of primary responses, consistent with real node morphology and functioning, are proposed. Anat Rec,