Histo Practice Exam 1

• *Describe the 3 main types of cells and the 3 main types of fibers found in connective tissue? Reference Table 5.6.

Studying these processes has contributed in understanding how the body communicates and works internally. Basic experiments have been done to illustrate these procedures as part of the biological curriculum. Such practical lessons are fundamental in learning biological sciences, which are significant in day-to-day life

What are the different levels of organization of a muscle down to myofilaments? What is a “sarcomere” and how are its proteins organized?

items presently in solid form in the lab kit or purchased separately for this laboratory

The tray lid was discarded and the dish containing membrane was put on the rocking platform for 15 minutes, washing the blot three times with 10 mL of TBS Tween at 5-minute intervals. Following the removal of all liquid from the dish at the end of the wash 10 mL of secondary antibody (goat anti-rabbit IgG HRP) diluted in milk blocking solution were added. Afterwards, the blot was incubated on the rocking platform for 20 minutes. During this incubation period the coomassie blue stained gel was placed on white paper for clear visibility and a photo was taken to document its appearance. This gel was previously incubated in Coomassie blue for approximately 1 hour. The gel was then discarded in the biohazard bag. After removing the membrane from the rocking platform the secondary antibody solution was removed from the membrane and discarded. Then 10 mL of TBS-Tween were added to the blot and the tray was manually shaken back and forth to remove leftover milk blocking solution and secondary antibody, which was immediately poured off. The blot was washed three times with 10 mL of TBS-Tween, at 5-minute intervals using the rocking platform. Upon completion of the washes a picture of the membrane was taken. All liquid was removed

In order for efficient fixation and processing times to occur, all tissues should be no thicker than 3mm (Washington School of Medicine, 2016). When a tissue sample is thicker than the recommended thickness, penetration and crosslinking of the sample tissue proteins will take an increased amount of time for this to occur. The centre of the tissue may undergo autolysis as the proteins located within the centre of the sample will not be reached at an optimum time. Additionally, the sample may be prevented from undergoing complete fixation by the chosen fixative. However when careful cutting techniques are utilised and the care is taken to cut optimally sized tissue samples, the desired fixation efficiency can be achieved. There are a few processes

A hemocytometer is a glass slide, known for its feature of having parallel and perpendicular lines which are used to count the number of cells (Kazam et al., 1991). The formula for cell viability was established by counting the number of cells that did not have Trypan Blue over the total number of cells. The cell density was established by using the microscope and the hemocytometer to count four large squares and dividing it by the total volume of the squares. After this was finished, the media was replaced with a different with a different solution that contained 2% DMEM adult horse serum, 1% penicillin/streptomycin and 2% amino acids. These were used in order to allow for cell differentiation. Dulbecco’s Modified Eagle Medium(DMEM), is a nutrient mixture that contains vitamins, glucose and amino acids that assist cells in differentiating. The cells on the plates were viewed with an inverted microscope and were then placed in the incubator for four

•Note: A formal lab report is not required for this activity. You may cut and paste this worksheet to a new Word document and adjust the spacing to fit your needs.

A cell suspension containing 0.5 -1.0 x 10^6 cells/ml in media 10% FBS (Fetal Bovine Serum) was placed on a 24- well plate so a monolayer can be created at the bottom of the wells. The first step will consist of the lab student aspirating the media from the wells making sure to disrupt the monolayer of cells at the bottom of the wells. Next the wells will be washed with PBS to remove dead cells and debris. After the wells are washed 1ml of media (DMEM) in a 5% concentration will be added to keep the cells from dehydrating. 5% Media is used because the goal here is to allow migration and minimize proliferation. Next the lab student will create a horizontal “scratch” along the bottom of the well using a P200 pipette. The lab student should use enough force to crate the scratch but not too much because too much force will create uneven scratches. Finally the treatments will be added to the wells. The plate will then be placed under the light microscope and pictures of the scratches will be taken which will be labeled as (T0) Time point zero. Other pictures will be taken 24 hrs. later at (T24) time point 24. The photos will then be analyzed and compared the lab group to determine if the is any cell

The antibody base method helps detect a specific protein in the sample being studied. IHC helps the researcher visualize the distribution and localization of certain cellular components in the cell. In the experiment, the tissue sections were thawed out for two hours and rehydrated in PBS for five minutes. Triton X-100 was then added to the PBS to help increase the permeability of the cell membrane. The day after, the tissue sections were washed with the PBS, and the sections were then incubated two hours with the secondary antibodies and the nucleic acid staining. The primary antibodies that were used are anti-connexin-43 mouse monoclonal, anti-α-smooth muscle actin mouse monoclonal, and an anti-α-actinin mouse monoclonal. The secondary antibodies that were used were Alexa-fluor antibodies (den Haan et al.,

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

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,

Lab Report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor.

When God created the world he knew the structure of the cell, he had every detail planned out. This shows how amazing and detail oriented our God truly is. In this lab we explored his creation more and looked at individual cells using microscopes. The microscopes were introduced in the first lab and were important to look at the cells more closely. While using either premade samples or the student’s cheek cells, the students learned how to use the microscopes well through adjusting the slide to find cells or focus the microscope so one could see the slides clearly. Through the microscope, the nucleus is visible because it is dyed darker than the other parts of the cell. This was true for all of the light microscope slides. The students were introduced to the inverted microscope containing mouse cells. The students

The stiffness I will use are 0.2kPa, 0.48kPa, 1.10kPa, 1.37kPa, and 1.67kPa to mimic the stiffness of matrices in vivo. To visualize the cells deforming the matrix, florescent microspheres will be incorporated into the matrix and traction force microscopy will be used to quantify the substrate deformation caused by the cells migrating on the 2D substrate. Ligand density will be varied by coating the polyacrylamide gels with type 1 rat tail collagen at varying concentrations 0.075 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.8 mg/ml. MMP activity will be varied using a broad spectrum MMP inhibitor marimastat at 5μM, 10μM, 15μM, 20μM and 25μM concentrations in PBS. Since the inhibitor is kept in a stock with DMSO I will also conducted a vehicle study to ensure the cells are not affected by the DMSO. Time-lapse images of the cells migrating on the substrate will be taken over 1hr 30min at 2 min increments using the florescent microscope available in Fraley Lab (figure 2). Cell migration will be quantified using metamorph software with the metavue track objects software. The data taken of x and y positions for each 1min 30sec time point will be used to find the average single cell velocity by finding the average of the distances traveled in each 1min 30sec time point. Invasion distance will found by finding the displacement of the cell from the difference between the x and y coordinates of the