Rat Brain Case Study

In this experiment, the primary antibodies against DAT were generated from the species Rattus norvegicus (the lab rat). The secondary antibodies (Chicken Anti-RatDAT) synthesized against Rat Anti-DAT, were generated from Gallus gallus domesticus (chickens). The Chicken Anti-RatDAT antibodies were synthesized by injecting certain purified rat antibodies into a chicken. As a result, a generation of the monoclonal antibodies Rat Anti-DAT, was essential to detect the DAT segment of the protein. Likewise, the polyclonal antibody Chicken Anti-RatDAT, linkage to the Fc portion of Rat Anti-DAT. Secondary antibodies are known to assist in the sorting, detection, and/or purification of target antigens by attaching to a primary antibody, that directly attaches to the target antigen.

1964). There was a statistically significant effect of time spent in the blocking solution and the fixative and staining quality. In future experiments, the time samples are placed in blocking solution and fixative should be monitored more closely and left for a sufficient amount of time to get better and more accurate results, concerning staining quality. On the contrary, there was a positive correlation between the amount of time the samples were left in the antibody solutions and staining results. The longer the samples were left in the antibody solution, the better the staining results were as attested by the statistical

Healthy Brain team at The Illinois Department of Human Services’ Division of Mental Health is pleased to present this proposal for your view. We look forward to partnering with you to provide help for mentally ill college students at Cook County, Illinois. The objective of healthy Brain programs is to promote and improve the quality of mental health care to college students. With growing national concerns regarding the prevalence and complexity of mental and behavioral health issues among college students and the negative impact these disorders have on the health and safety of higher education communities, student retention, student learning/ academic progress, and the human potential of students.

Second, for the western blotting, the primary antibodies that used to recognize the protein of interest (FLAG-PTEN) are rabbit anti-PTEN antibodies and the secondary antibodies that bind to the primary antibodies and convalently linked substrate to produce visible signal are goat anti-rabbit conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used to immobilize protein of interest are anti-FLAG monoclonal antibodies from morine cell. As introduction above, those antibodies come from different species. In order to be successful in western blotting, the primary antibodies recognize the protein of interest as well as bind to it. Consequently, rabbit anti-PTEN antibodies were used to bind to FLAG-PTEN. Meanwhile, goat anti-rabbit conjugated HRP antibodies, the secondary antibodies,

In the beginning, the cells were centrifuged for 5 minutes at 300xg then supernatant was eliminate. Then, the remaining pellet was ease with flicked the tube. After that, 350 µL Buffer RTL was added and mixed by pipette. The lysate of cells were homogenized by pipette for 15 seconds. After the homogenization, 70 % ethanol in 350 µL was added into the lysate and mixed by pipette. Then, samples were transfer to Rneasy spin column and placed into

Total proteins were prepared from cells of embryonic cortex at different ages (E10–18) and adult brain (2 months) with 500 µl of cell lysis buffer. Cell lysis buffer contains 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM PMSF. After homogenization on ice for 10 min, the supernatant of each sample was collected and stored in aliquots at -80 °C. For each sample, the protein concentration was determined by Bradford assay (Thermo Fisher Scientific). Cell lysate (20–50 µg) was resolved on 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF or nitrocellulose membranes. The protein blots were blocked with 5% milk in TBS (10 mM Tris–HCl, pH 8.0, 150 mM NaCl) overnight at 4 °C and incubated with primary antibodies at a dilution of 1 to 1000–2000 in 5% milk prepared in TBS. The secondary antibody was donkey anti-mouse IgG-700 or anti-rabbit IgG-800 (LI-COR). Quantitative western blot was detected by the Odyseey CLx Infrared Imaging System according to the company’s instructions

Firstly the wax was removed by putting the 4 slides containing mouse spleen and thymus on hot plate to melt the wax. Secondly the slides were rehydrated by placeing the slides into alcohol starting from absulote alcohol 100 % to 70%. Leaving each slide for 2mins. The 4 slide sections are placed in Ehrlish’s Haematoxylin (30 mins) to regressive stain. Slides were removed from staining and rinsed with clean water (30s). then the Slides were placed into acid alcohol (3-4s). sections changed from blue to red and then after slides were placed into ammoniated alcohol until turned blue,

On our SDS-PAGE, our protein standards were separated alongside our protein samples which allowed determination of the sizes of our protein in our sample. SDS denature proteins and coats them with a negative charge allowing for their electrophoretic separation by polyacrylamide gel electrophoresis (PAGE). As you can see, after being stained with the coomassie blue, we could detect proteins of various sizes and our unknown protein expressing GST was detected in our Pure B sample (Figure 2). The distance travelled from the wells on the SDS-PAGES by our protein standards were measured and compared to our protein ladder in Daltons (Table 2), from which we could construct our semi log plot. On the semi-log plot, our unknown protein within our Pure B travelled 27mm from the well with a size of 29,000 Dalton (Figure 3). To identify the specific protein in the gel, the western blotting method was used to detect which of the cultures (A or B) contains GST (Figure 4). After the cassette was taken out of the electrophoresis unit, colors of our protein standards appeared on top of our membrane surface. After blocking, primary antibody incubation, and secondary antibody incubation, we could clearly identify our GST in our Pure B and Crude B (Figure

In the fluorescence experiment, the anti-GFAP (Glial fibrillary acidic protein) antibody binds to the GFAP protein in the tissue. Putting on the secondary antibody helps visualise where the GFAP is and acts as an amplifier. The second antibody has an added fluorescent probe and will glow when an electromagnetic energy of a certain wavelength shines on it. This will help see where the GFAP is bound and the type of cell it is in. In this case, in the brain tissue, will be

Moreover, further deletion mapping could be performed to narrow down the specific sequence within the third SIRPT of sacsin that binds JIP3.

The plate was then read for absorbance at 540nm using the microplate reader. The neutral red assay was chosen because it assesses the lysosomal integrity of cells. As the integrity of the lysosome in cells is dependent upon a very wide range of healthy internal machinery, as opposed to a single or a few individual pathways, it provides a good overview of general cellular health as deficient systems inside the cell are likely to impact on the cells ability to retain the neutralred stain [19] .

15 cell lines were acquired for the purpose of this experiment. Process of subcellular compartment fractionation was performed on these cell lines, this included complete cells, nucleus and cytosol (seen in Table 1).

The brain has four main structures; the Cerebrum, Cerebellum, Limbic System, and the Brain Stem. The Cerebrum, also known as the Cerebral Cortex, is the largest part of the human brain. It is divided into four parts that are called “lobes”: there 's the frontal lobe which is linked with reasoning, planning, parts of speech, emotions, and problem solving- the parietal lobe which is linked to movement, orientation, and recognitions- the occipital lobe which is linked to visual processing like object and spatial recognition, and the fourth one which is the temporal lobe that is linked to the perception and recognition of memory, speech, and sounds. The Cerebellum, also known as the “little brain”, is linked to coordination, movement, posture, and balance. The limbic system usually referred to as the “emotional brain” is located inside the cerebrum and it is tied onto our emotions and more importantly, our survival mode. Lastly, there is the brain stem that is in charge of basic life functions like breathings, our heart beating, and blood pressure. All of these four brain functions have key important roles on our everyday lives, but is there anything that can affect them? The brain happens to actually be affected by many things, either from accidents, or emotional pain that leads to depression, the brain grows differently depending on its external stimuli, and a great example of this would be music.

In a parallel step, image texture features are extracted deriving the other part of the feature vector. Five texture features

The cells were re-suspended in lysis buffer (7 M urea, 2 M thioura, 4 % CHAPS, 40 mM tris, 0.2 % Biolyte 3/10, and 50 mM DTT). Whole-cell extracts were prepared by sonication of cell suspension for 4×40 s of 30 kHz with 20 s intervals followed by centrifugation at 14,000×g for 15 min at 4 °C. Protein concentration was determined by Bradford method. The lysate aliquoted and kept in −80 °C for further use.