Histological Study : Light Microscope Examination

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.

b. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products. After an incubation

The three tissues being analyzed in this experiment, those of the kidney, heart and liver, were taken from the animal Bos taurus. The tissue homogenates used were made by adding 1 gram of tissue to 20 ml of sucrose phosphate buffer. The buffer was composed of 250 mM sucrose, 50 mM NaPO4, with a pH of 7.4. This mixture was homogenized with a high-speed blender for 3 pulses for a duration of 10 seconds each. The homogenates were then filtered through a cheesecloth and stored at -70° C.

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

While browsing the well-known career site Monster, I discovered a listing for a histology technician position in Greensburg. The career opportunity is with Excela Health, who has a respectable reputation in the area. In addition to the suitable location, the company also offers a competitive benefits package and financial well being and security of the organization.

A horseradish peroxidase (HRP) is then added to each of the microtiter well which leads to the formation of a sandwich. The sandwich is composed of a polyclonal antibody, monoclonal antibody and chromogranin A. The unbound monoclonal antibody is later on washed out in the subsequent washing step. Detection of this immunocomplex is achieved by incubating the well with a substrate solution in a timed reaction and then a spectrophotometric microplate reader is used to take the readings (Stivanello

The first experiment we ran was the α-smooth muscle actin (asma) stain. The purpose of this stain was to use a primary and secondary antibody to indirectly stain the microfilaments that characterize myofibroblasts. Under a fluorescent microscope, this will provide a visual representation of how many fibroblasts in our samples had differentiated into myofibroblasts. To set up for this experiment, fibroblasts were plated on coverslips and incubated for seven days, to reach 100% confluency, in either the presence or absence of allopurinol. After one week the coverslips were fixed with methanol and the first antibody, a mouse anti-human asma, was applied onto the coverslip and kept refrigerated overnight. The next day the coverslips were rinsed with PBS, all rinses are performed with PBS, and the second antibody, a goat anti-mouse rhodamine, was applied to the coverslips for thirty minutes in a dark place. Lastly, after another rinse, a DAPI counterstain, which would interact with the DNA in the nucleus of every fibroblast to make them visible, was applied for fifteen minutes and rinsed. Then the coverslips were mounted onto slides with glycerol and looked at under the fluorescent microscope. The nuclei of all of the cells lit up blue and the microfilaments in the differentiated fibroblasts, myofibroblasts, lit up bright

• Chapter 1 1. Anatomy and physiology are both considered to be branches of biology that work together to explain the structure of our body and its ability to function. 2. Cytology deals with the study and function of cells which make up the human body. 3.

detailed image of the kidneys and other organs, a kidney biopsy where a small piece of

The recognition of a well-defined ANA staining pattern using the HEp-2 cell substrate may be helpful in

The aim of this experiment was to utilise PCNA and staining procedures to determine whether different tissue cell samples proliferate. Tissue which exhibited brown stains prove cell proliferation and mitotic divisions, this would suggest it being the in the first group of Bizzozero’s grouping of tissue types. Tissue samples which showed no brown staining meant no cells had proliferated and therefore were of a different tissue type that do not regenerate while present in the body. The PCNA protein is present during cell division/mitosis therefore, its presence suggests that cell proliferation is occurring or has occurred. An area can be stained using brown diaminobenzidine (DAB) by using an antibody that is against the PCNA protein. A primary antibody then attaches to the PCNA molecule, and a secondary antibody (horse radish peroxidase) attaches to the primary antibody. A

The hippocampus was rapidly collected and homogenized in Cell lysis buffer (beyotime, P0013, China) containing protease and phosphatase inhibitors. The homogenate was centrifuge at 10,000rpm/min for 5min at 4 ̊C. The protein concentration of the supernatant was determined by SDS-PAGE with standard protein. Then the sample was run by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membrane. The membranes were incubated with blocked solution (5% nonfat milk in PBS) at room temperature for 1 h and probed with primary antibody against CaMkII (Cell signaling, #12716) and PSD95 (Abcam, ab192757) at 4 ̊C overnight, then the membranes were incubated with horse radish peroxidase conjugated secondary antibodies for 1 h at room

This “diagnostic dilemma” may bring on consequences for the patient, as histologic variations of AOTs presenting similar features to other odontogenic tumors or cysts may have unspecific clinical and radiographic features that overlap with those of other odontogenic tumors, which reinforces the fundamental role of microscopic examination in diagnosis to ensure appropriate treatment.

Briefly, antiserum raised against the rtBbigAMA-1 was applied as the first antibody (1: 200) on the fixed smears and incubated at 37 º C for 1 h in a moist chamber. After washing with PBS Tween 20 (PBST) three times, Alexa-Fluor® 488-conjugated goat anti-mouse immunoglobin G (IgG) (Molecular Probes, Dallas, Texas, USA) was applied as a secondary antibody (1:200) and then incubated at 37 ºC for 30 min. The slides were washed three times with PBST and incubated with 2.5

For generating antigen specific antibodies, human peripheral blood mononuclear cells (PBMC) were isolated from three healthy volunteers. PBMC were then immunized with interleukins and BoNT. Invitro immunized cells were then isolated and RNA was extracted from it. The RNA was then reverse transcribed to form cDNA. The cDNA was then used for template for production of human IgG Fd (consisting heavy chain variable and constant region) and light chain fragments using specific primers. The DNA obtained by amplifying IgG Fd and light chain was digested with respective digestion enzymes and ligated into different lambda phage and cloned. The cloned lamda phage DNA was then separated by 0.8% Agarose Gel Electrophoresis. The purified gel fragments of