This combined masters can answer key questions in the field of epididymal research, such as what protein targets are involved in the regulation of sperm maturation and storage. The main advantage of epididymal isolation technique is that it also applicable to isolate a similar population from other regions of epidermis among different species. To begin this procedure, transfer the epididymal paraffin sections to a fume hood.
Add enough xylene to the slide jar to completely immerse the tissue section. Rehydrate the tissue sections by immersing them in graded ethynol solutions diluted in purified water as outlined in the text protocol. Wash the sections in a slide jar for five minutes with sufficient PBS to completely immerse the entire tissue section.
Next, decant the appropriate antigen retrieval solution into a slide rack. Microwave the solution until it is boiling. Completely immerse the slides in the solution, subjecting the the tissue sections to heat-induced antigen retrieval solutions.
After this, remove the slide container from the microwave and let it cool to room temperature. Rinse the slides in a jar containing PBS for five minutes. Using a liquid-repellent slide marker pen, trace around the tissue section.
Transfer the slides to a humidified container. Apply blocking solution for one hour at 37 degrees Celsius. Then, rinse the slides once with PBS.
Incubate the sections with an appropriate primary antibody diluted to an experimentally-optimized concentration in filtered 1%BSA PBS at four degrees Celsius overnight. After re-warming the slides to room temperature, wash the slides with PBS on a shaking platform at 60 RPM for 10 minutes. Repeat this wash two additional times.
Incubate the sections with an appropriate secondary antibody diluted in filtered 1%BSA PBS at 37 degrees Celsius for one hour. From here onward, keep the slides in the dark. Wash the slides three times with PBS on a shaking platform at 60 RPM for 10 minutes per wash.
Then, stain, mount and seal the samples as outlined in the text protocol. Immediately after euthanizing the adult mice, profuse their vasculature with PBS that has been pre-warmed to 37 degrees Celsius to minimize the blood contamination of epididymal tissue. Carefully dissect the epididymis free of overlying fat and connective tissue, and rinse with modified BWW medium to reduce any potential for surface blood contamination.
Blot the epididymal tissue to remove excess media and dissect the caput epididymis. Then, transfer the dissected caput epididymis to a fresh petri dish containing BWW medium, making sure that the amount of medium is sufficient for the final recovery. Using a razor blade, make a number of small incisions into the caput tissue.
Do not mince the tissue. Incubate at 37 degrees Celsius with mild agitation for 30 minutes to release the luminal contents. Using 70 micrometer membranes, filter the resultant suspension and remove the cellular debris.
Collect the suspension and subject it to successive centrifugation steps at four degrees Celsius with increasing velocity to eliminate cellular debris. Next, prepare discontinuous iodixanol gradients as outlined in the text protocol. Prepare the gradient in an ultracentrifuge tube using each fraction of 450 microlitres.
Visually inspect the gradient after each fraction is applied to ensure that the interfaces are successfully formed between each layer. Carefully add 450 microlitres of epididymal luminal fluid suspension on top of a single gradient. Ultracentrifuge the gradients at 160, 000 times g and four degrees Celsius for 18 hours.
Gently remove 12 equal fractions from the centrifuged gradient, starting from the uppermost layer and progressing toward the bottom of the gradient. If applicable, pool the equivalent fractions recovered from each gradient. After fractions nine through 11 have been recovered and pooled, dilute them into two milliliters of PBS.
Ultracentrifuge the samples at 100, 000 times g and four degrees Celsius for three hours to pellet the epididymisms. Then, carefully aspirate and discard the supernatant, making sure to not disturb the epididymism pellet. It is essential to assess epididymism purity before any downstream applications.
After preparing the sterile coverslips, passage aliquots of 200, 000 mEcap 18 cells in each well of the 12 well plate containing the coverslips. Culture the cells as outlined in the text protocol. Once the cells have adhered to the coverslip, discard the medium and rinse the cells twice with PBS.
Add a sufficient amount of 4%paraformaldehyde to immerse the entire coverslip, and fix the cells at room temperature for 15 minutes. Then, discard the paraformaldehyde solution and rinse the coverslips twice in PBS. Immerse the rinsed coverslips in 0.1 Triton X-100 in PBS for 10 minutes to permeabalize the cells.
Rinse, block and immunolabel the cells as outlined in the text protocol. First, passage aliquots of mEcap 18 cells into a 6 well plate as outlined in the text protocol. Once adhered, wash the cells three times with FBS-free mEcap 18 cell medium to remove residual FBS and any associated protein contaminants.
Then, add 1.5 milliliters of FBS-free mEcap 18 cell medium to each well. Incubate for 12 hours in a 37 degree Celsius incubator under an atmosphere of 5%co2. After this, collect the cell medium and centrifuge at 2, 000 times g for 10 minutes to remove all cellular debris.
Using a standard trypan blue exclusion assay, assess the mEcap cell viability. Add 20%volume of chilled 100%trichloroacetic acid to 80%volume of condition cell medium to precipitate the proteins released from the cultured mEcap 18 cells. Incubate at four degrees Celsius overnight with constant mixing.
Next, centrifuge at 17, 000 times g and four degrees Celsius for 10 minutes. Discard the supernatant and wash the protein pellet twice with chilled acetone. Then, centrifuge again at 17, 000 times g and four degrees Celsius for 10 minutes.
Carefully remove and discard the supernatant once more. Then, air dry the samples in the fume hood to remove any residual acetone. Each of the DNMI isoforms investigated displays a distinct immunofluorescence localization profile.
DNMI is characterized by relatively modest diffuse labeling of the epididymal cells throughout the initial segment and caput epididymis. The DNM2 isoform is first detected in the vicinity of the opposing basal and apical border of cells in the initial segment, before repositioning to the supranuclear domain in cells within the adjacent downstream caput segment. The intensity of this labeling gradually decreases between zones two and five of the caput epididymis, which essentially mirrors the secretory activity of these epididymal segments.
The supranuclear labeling of DNM2 is subsequently seen to correspond to the distribution of the golgi apparatus within caput principal cells. The DNM3 isoform is mainly detected in the apical domain of a small number of caput epithelial cells, which are seen to correspond to the clear cell subpopulation by co-labeling with the recognized clear cell marker ATP6V1B1. Next, an immortalized mEcap 18 cell line is utilized as an in vitro model to study epididymal cells'secretory activity.
The distribution patterns are seen to be consistent with those seen in caput epididymal tissue sections, with DNM1 detected throughout the cytoplasm of mEcap 18 cells, DNM2 concentrated within the supranuclear of these cells, and DNM3 characterized by discrete foci of membrane staining within a small sub-population number. While attempting the immunofluorescence detection, it's important to remember to optimize the conditions for each antigen. The epididymal zone population's isolated are readily amenable for use in downstream applications including co-incubation with spermatozoa and ocalgo per filing analysis both of which are powerful approaches for enhancing our understanding of the role of epididymosome in regulating epididymal sperm maturation.
