This method can help answer key questions in the post-natal development field about the way milk suckling influences stress signaling in the brain. This technique can be used to capture specific brain areas during the first natural starvation period and to compare them to respective areas shortly after the first natural colostrum feed. Understanding how suckling influences the physiology of behavior and emotion enables the use of an antagonist to various receptors during ex-vivo brain tissue analysis.
To obtain the nuclei punches, remove the rat pups by their tail with a gloved hand. After harvesting the brain, rapidly place the whole organ in a polymethyl methacrylate brain mold at room temperature and immediately use a new razor blade to make 500 micrometer thick slices of the tissue. Lay the slices rostral to caudal in a Petri dish as they are obtained to maintain the correct orientation of the sections.
When the entire brain has been sectioned, quickly add artificial cerebral spinal fluid without glucose to the dish and incubate the slices for 60 minutes at 28 to 30 degrees Celsius with constant stirring on an orbital shaker. Use a brain atlas and anatomic landmarks on each tissue to identify the brain nuclei to be punched and place the slice with the nuclei of interest in a new Petri dish under a dissecting microscope. Once visualized, use a coring tool to quickly punch out four to six different nuclei and rapidly immerse each punched nucleus in 0.06 mL of ice cold protein extraction buffer in the appropriately labeled micro-centrifuge tube containing proteus inhibitors and phosphatase inhibitors for 60 minutes.
At the end of the incubation, centrifuge the extracted nuclei in a cooled mini-centrifuge, and use a micro-pipette to carefully transfer 0.055 mL of supernatant from each tube into the appropriate pre-cooled 1.5 mL micro-centrifuge tube on ice. Before negative 20 degrees Celsius storage, transfer 0.012 mL of supernatant from each protein stock tube into the appropriate 0.5 mL pre-cooled tube on ice for the first sample preparation. To prepare the samples for in-capillary protein measurement, add 0.003 mL of master mix reagent from a protein lysis kit to each of the samples in the 0.5 mL labeled tubes.
Next, add 0.004 mL of master mix reagent to a biotinylated molecular weight ladder solution in 0.016 mL of deionized water, and denature the ladder and protein extract samples in a 95 degrees Celsius heat block, storing all of the tubes at four degrees Celsius after five minutes. For signal protein analysis, add 0.01 mL of freshly prepared luminol peroxide solution to wells E-1 to E-25 of a new automated Western plate, 0.01 mL of secondary anti-mouse antibody to wells D-2 to D-25, and add 0.01 mL of streptavidin horseradish peroxidase from the kit to well D-1. Add 0.01 mL of freshly prepared primary antibody into wells C-2 to C-25, and 0.01 mL of antibody diluent two solution to wells C-1 and B-1 to B-25.
Leave the row F wells empty and fill the five compartments of the three rows below the F row with 0.45 mL of wash buffer. Next, briefly spin the refrigerated samples for 30 seconds and add 0.03 mL of each sample to each well of row A, starting with A-2. Then, add 0.005 mL of the biotinylated molecular weight ladder to well A-1 and cover the plate for centrifugation.
While the plate is centrifuged, open a new run file in the automated Western associated software and initiate a molecular size assay. In the assay page, enter the sample names into each capillary as well as the names of the primary and secondary antibodies. At the end of the centrifugation, place the plate in the automated Western instrument and peel the cover from the capillary cartridge box.
Following centrifugation, don't forget to inspect for air bubbles, because air bubbles in the capillary can block the electrophoretic current and fail the entire round. Insert the cartridge into the designated position within the instrument and close the door. Click start.
When prompted with the type of the assay, enter the name of the experiment into the appropriate text box and click okay. When prompted by the activated run date and the ID number on the run file, make a note of the time when the run ends. At the end of the run, discard the capillary cartridge into the sharps disposal and the plate into the biohazardous materials disposal.
After the electrophoresis separation, check the run files for immune reactivity peaks of antigens at 40 to 43 kilodaltons which stand for phosphorylated eukaryotic translation initiation factor 2A. Where molecular weight peaks of this size are missing, right click below the curve and select add molecular weight to peak to ensure that the sizes and arbitrary quantities below the curve are recorded. Within the unprimeds tissue samples, binding amino globulin protein levels in the nuclei of the solitary track are significantly higher compared to all of the other regions.
Priming of the gut tissue with colostrum decreases binding amino globulin protein levels in the nuclei of the solitary track and has no effect on paraventricular nuclei and super-optic nuclei. Conversely, priming increases binding amino globulin protein levels within the cortex, striation nuclei, and medial preoptic nuclei relative to the unprimed tissue, implying that binding amino globulin protein levels in the various tested brain nuclei respond differently to gut priming and there is not yet any demonstrated crosstalk between the various nuclei tested. Eukaryotic translation initiation factor 2A and phosphorylated eukaryotic translation initiation factor 2A levels are elevated in the unprimed condition relative to other nuclei.
After priming, levels of both eukaryotic translation initiation factor 2A and phosphorylated eukaryotic translation initiation factor 2A are reduced relative to nuclei of the solitary track compared to unprimed tissue. In all other tested nuclei, priming increases the levels of eukaryotic translation initiation factor 2A and phosphorylated eukaryotic translation initiation factor 2A relative to unprimed tissue. However, levels of phosphorylated protein kinase receptor are low in unprimed samples relative to primed samples in nuclei of the solitary track, strongly suggesting that another kinase is involved in the phosphorylation of eukaryotic translation initiation factor 2A.
After its development, this technique paved the way for researchers in the field of post-natal development to explore the signaling pathways involved in tissue growth and differentiation and whether they are influenced by or independent of suckling. Don't forget that working with volatile reducing agents like, for example, dithiothreitol, can be hazardous, and precautions such as preparation in the chemical hood should always be taken when preparing this procedure.
