Lab Protocols

Apoptosis: Programmed cell death (PCD) MATERIALS : 1. 1 X PBS (PBSAz, 1 X PBS, e.g., Irvine Scientific, CA, containing 2% newborn calf serum and 0.1% sodium azide) 2. 7-Amino-actinomycin D (7-AAD, e.g., Calbiochem, CA) 3. Human AB serum, heat-inactivated (HAB, e.g., Irvine Scientific, CA) 4. Refrigerated centrifuge Preparation of 7-AAD stock solution Dissolve 7-AAD powder (1mg) first in 50 microliters of absolute methanol, then add 950 microliters of 1 X PBS. Final concentration is 1mg/ml. Keep solution at 4 ° C protected from light. Solution can be stored for at least up to 6 months. METHOD : Principle: Apoptotic cells, probably due to a change in membrane permeability, take up some 7-AAD and become 7-AAD dim compared to live cells which remain 7-AAD-. Late apoptotic or necrotic cells which have lost membrane integrity appear 7-AAD bright . Reference : Schmid I, Uittenbogaart CH, Keld B, and Giorgi JV: A rapid method for measuring apoptosis and dual-color immunofluo...

Materials Microscope Prepared slide with three colored, crossed threads Procedure Place a slide containing three colored and crossed threads on the microscope stage. Determine the width (diameter) of the threads using the procedures from Exercise 1.3 . Locate a spot where all three threads cross each other at the same point. Use the fine focus control to focus first on the lowermost thread, then the middle thread and finally, the uppermost thread. List the order of the threads from the top to the bottom, by indicating their color. Focus on the top of the uppermost thread. Note the scale markings on the fine focus knob and record the calibrated reading directly from the fine focus control. Carefully rotate the fine focus and stop when the microscope is just focused on the upper edge of the next thread. Record the reading from the focus control below. The difference between the two readings is the depth (thickness) of the upper thread. Position Color Diameter ...

Figure 1.7 Superimposed ocular and stage micrometers Materials Microscope Ocular micrometer Stage micrometer Millimeter ruler Prepared slide with letter e Procedure Place a stage micrometer on the microscope stage, and using the lowest magnification (4X), focus on the grid of the stage micrometer. Rotate the ocular micrometer by turning the appropriate eyepiece. Move the stage until you superimpose the lines of the ocular micrometer upon those of the stage micrometer. With the lines of the two micrometers coinciding at one end of the field, count the spaces of each micrometer to a point at which the lines of the micrometers coincide again ( Figure 1.7 ). Since each division of the stage micrometer measures 10 micrometers, and since you know how many ocular divisions are equivalent to one stage division, you can now calculate the number of micrometers in each space of the ocular scale. Repeat for 10X and 40X, and 100X. Record your calculations below. Microscop...

Materials * Microscope equipped with 100X, oil immersion lens * Immersion oil * Prepared slide of bacteria or suitably small objects Procedure 1. Place the prepared slide on the microscope stage. Using the 10X objective, focus the microscope on an appropriate field containing bacteria. Center the bacteria in the field of view, by manipulating the lateral movement knobs. 2. Rotate the nosepiece to the 40X objective and refocus with the fine focus. Again, center the object which you wish to examine. 3. Rotate the nosepiece so that an intermediate position between the 40X and 100X objectives is obtained. 4. Place a small drop of immersion oil 10 on the center of the viewing area of the slide. 5. Continue to rotate the nosepiece so that the 100X objective is rotated into the oil. Do not under any circumstances place the 40x objective in the oil Use only the fine focus and refocus your specimen. 6. Determine the shapes of the b...

LEVEL I Figure 1.6 Nikon S-Cb Figure 1.6a Nikon Binocular Microscope Refer to Figure 1.6 and Figure 1.6a for the names of the various components of a Nikon binocular microscope. Materials Binocular Microscope 5 Microscope slide with letter e Procedure Pick up a microscope from the cabinet by placing one hand under the base and the other on the arm of the microscope. Most microscope damage is due to careless transport. It is important that you carry the microscope securely, with two hands, and in an upright position. Remember that you are handling $1,000 of precision instrumentation. Place the microscope in front of you, unwind the power cord and plug it in. The microscope is normally provided in its storage position, that is, with its eyepieces pointed back over the arm. This takes less room in a cabinet, but is not the position for which it was designed to be used. If your instructor approves, slightly loosen the screw holding the binocular head and rotat...

Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis. Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane, Thomas Brunner, Ella Bossy-Wetzel and Douglas R. Green Details of this protocol, Acridine Orange/Ethidium Bromide (AO/EB) Staining To Detect Apoptosis (Subscription Required), are located on a web site other than Biocompare Protocols. Source Link : http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4493