Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-ß-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Tissue-specific cross-reactivity of connexin32 antibodies: problems and solutions unique to the central nervous system.

Gap junction proteins are a highly homologous family of 21 connexins. Here, the authors describe a tissue-specific technical artifact complicating analysis of connexin32 protein expression in the central nervous system. The authors show that in brain, but not liver, eight commonly employed antibodies exhibit a higher affinity for a cross-reactive protein that masks the detection of connexin32. Cross-reactivity is evident in Western blot analyses when proteins are subjected to reducing/denaturing conditions but not immunoprecipitation or immunofluorescent applications. Through bioinformatic analyses, tested by sucrose gradient fractionation and immunoblotting of lysates from connexin null-mutant mice, the authors show that the cross-reactive protein is not found in the same cellular compartments as connexin32 and is likely not a member of the connexin family. These findings are presented with the intent of helping to reduce the amount of time laboratories currently expend in validating changes in connexin32 expression in the central nervous system.