Histological Study : Light Microscope Examination

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

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.

The protein/DNA ratio indicated a greater ratio in the heart homogenate in comparison to the liver and kidney. The relative cell size in the heart homogenate could be inferred to be much greater because of such a high ratio. (Table 1)

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.

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.

The filter flask, vacuum, and SNAP i.d. 2.0 were securely connected with tubing. The proteins were transferred to a membrane and was incubated with primary antibody. The primary antibody was then washed three times. Afterwards a secondary antibody was added and the same procedure was repeated. 10mL of 5-bromo-4-chloro-3-indoyl phosphate/ nitro blue tetrazolium chloride chromogenic substrate solution was added to the blot which was then wrapped with aluminum foil and labeled.

diH20 acted as a negative control in the experiment, all cells, both in intrafollicular zone and in interfollicular zone stained purple. Cells stained brown in the negative control indicate non-specific staining.

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

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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.

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

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

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