Posts

Showing posts from 2011

Zebrafish Care and Experimental Techniques: In Situ Hybridization - Zebrafish Sections

Image
**Day 0: Goals: To preserve zebrafish and obtain sections using the cryostat. **Everything used on this day needs to be RNase free . Wear gloves and be sure to use only PBS that has been treated with DEPC. 1. Anesthetize zebrafish 2. Fix in freshly thawed BT fix for 3 hours at room temperature. Keep solution on the "belly dancer" so that it is constantly moving. 3. Rinse 3 x 5 minutes in 1X PBS. Leave embryos in pbs until you are ready to section them. Embryos can be stored in the fridge until sectioning, but keep the days between fixing, sectioning and staining as short as possible. 4. Rinse fish in 30% sucrose and PBS until embryos sink approx. 30mins. 5. Trnsfer embryos from sucrose to plastic molds, 5 fish pre mold, with heads facing the side with writing. Absorb as much sucrose as possible. 6. Add OCT to the mold, and swirl fish until they are oriented correctly. Freeze in -80 degree freezer. 7. Follow instructions for sectioning using the cy

Zebrafish Care and Experimental Techniques: Real Time PCR

Materials: -primers (24 and 25 are for slitrk 3) -cDNA (40 hour/90 hour) -10mM dNTP (nucleotides) -buffer 5x -water -enzyme (cooler in freezer) Combine in a PCR tube (50 microliters of solution): 1. 35.5 uL of water 2. 10uL 5x buffer 2. 1uL of JR 24 3. 1uL of JR 25 4. 1uL of dNTP 5.1uL of cDNA 6. 0.5 uL of Fusion hot start enzyme Add water to the PCR tube first. Make sure you use the PCR tubes because they fit in the machine. After all contents have been added, flick PCR tube and centrifuge for 4 seconds. To Run the PCR Machine: The power switch is on the back. The lid will click, and then the wheel should be twisted until you feel resistance. Program: JRRTPCR enable heated lid The reaction takes several hours. The machine with have a block temp of 4 degrees when done. Remember to turn off the machine when finished using it.

Zebrafish Care and Experimental Techniques: Midi-Prep of Plasmid DNA

Day 1: see "Bacterial Transformation by Heat Shock" **Day 2: Starter Cultures and Inoculate into Large Bacteria Flasks** NOTE: Starter culture is preferable, but not required; can immediately go to larger flasks if you want to STARTER CULTURE: 1. Aliquot 2ml of the LB+Amp solution (0.02g Amp/200ml LB) into 14ml round bottom tubes 2. Using a yellow pipet tip, pick up an isolated large colony and drop the tip into the correct tube 3. Cap and place into incubator (37C) with shaker set to 200rpm for ~6 hours Note: At this point, can refrigerate starter cultures until ready to inoculate and do midi-prep kit INOCULATION: 1) Add Amp to LB broth at 100ug/ml (so, for 500ml of LB broth, add .05g Amp) 2) Add 100ml of LB+Amp into a 1L flask for every plasmid you are inoculating 3) Transfer 200ul of bacteria from starter culture into corresponding flask 4) Cover each 1L flask with aluminum foil 5) Incubate for 12-16 hrs in Dana Incubator (if necessary, use paper towels t

Zebrafish Care and Experimental Techniques: Gel Electrophoresis

Materials: -All materials are EtBr exposed, so wear gloves! -Materials are kept on a tray on the lab bench 1. TAE solution 2. Agarose 3. EtBr 4. Plate set-up 5. DNA ladder (in fridge) 6. PCR product For 70mls of 1% gel solution: 1. Add 70 mls of 1X TAE to the flask 2. Add 0.7g of agarose to the flask 3. Microwave the solution for 1:30 mins, watching for bubbles 4. Microwave again until the solution clears and bubbles but does not overflow 5. Allow the solution to cool to 50 degrees celsius (warm to touch) 5. Under the hood, add 3.5uL of EtBr to the solution (before it cools completely!) 5. Stir the solution gently so that the EtBr dissolves 6. Once dissolved, pour the solution onto the gel plate set-up. Make sure the comb is properly oriented and that the screws are tightened. 7. Allow plate to cool for at least 25 mins 8. Place plate into buffer (TAE) and gently remove the comb 9. Add enough TAE to cover the wells 10. Use a DNA ladder, 20 uL in one well 11. Sp

Multichannel Images with Bright-field in Nikon Elements

To take a multichannel image in Elements: 1. click acquire --> capture multi channel image --> manually 2. There should now be an open window with tabs along the bottom for each of the image channels 3. Bright-field should be included in those tabs 4. If Bright-field is not included, you will need to add a channel. a. click acquire --> capture multichannel image --> multichannel setup b. click on an unchecked black box to add a channel, assign the channel the appropriate settings c. for Bright-field set the comp. color to Bright-field d. when you close re-open the multichannel image you should have a bright-field tab 5. Click the tab along the bottom that corresponds with the image you want to take 6. set-up the image using the microscope or i-control 7. click capture when you are ready to take the picture 8. Take all of the images you need to compile and then click the four box icon in the left hand corner. The icon is a shaded box with three colored boxes p

Calcium phosphate transfection protocol - Protocol for cell transfection using the calcium phosphate method

Procedure: Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Add 40 μl per well immediately after plating (20 μl each of the luciferase plasmid with 20 μl of the beta gal mix). All mixing (except that which requires vortexing) should be done in the sterile cell culture hood. Total volume 400 μl 600 μl 800 μl Sterile 2.5 M CaCl 2 25 μl 37.5 μl 50 μl Plasmid 10 μg 15 μg 20 μl Sterile H 2 0 Total of 200 μl when added to above. Bring to total of 300 μl with sterile H 2 0 Bring total to 400 μl with H 2 0  Combine the above three reagents in a hood using a sterile 1.5 ml Eppendorf tube. Mix gently. Do not vortex. Sterile 2X HBS (hepes buffered saline

Mouse adipocyte glucose uptake assay - Protocol for glucose uptake assay in mouse adipocytes

Procedure Day 0 cell culture: Grow 3T3-L1 cells in high glucose DMEM + 10% Calf Serum + P/S/F in 6-well plates. After cell have reached complete confluent, replace growth media with stimulation media containing: High glucose DMEM: 10% FCS, P/S/F 1 µl / ml of Insulin (10 mg/ml, 1000x in HEPES, final 10 µg/ml) 1 µl / ml IBMX (3-isobutyl-1-methylxanthine, 500 mM stock, 1000x), final 0.5 mM 0.1 µl / ml Dexamethasone (10000X in ethanol, final 1 µM). Day 3 Replace with fresh media containing Insulin only, no IBMX, no Dexamethasone.  Change the media on day 6.  Cells will be differentiated on day 7-9. Day 6 Cells will be cultured in serum-free DMEM for 2 hr ± treatments Incubate cells  in 1 ml of KRH with 100 nM insulin for 20 min ± treatments. Add 1 µCi of 2-deoxy-D-[2,6-3H]-glucose (50 µl in volume) to each well for 10 min. Wash cells with KRH buffer 3X. Lyse cells in 1 ml of RIPA buffer.  Count 300 µl. KRH buffer: 25 mM HEPES-NaOH [pH 7.4] 120 mM NaCl 5 mM KCl 1.2 mM

Cardiomyocyte immunofluorescence protocol - Protocol for glucose uptake assay in mouse adipocytes

Image
Procedure describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes View a pdf version of this page   Anti-sarcomeric – α actinin and ANP Staining Fix cells as usual in 3.7% paraformaldehyde in PBS. Rinse cells twice with PBS after fixation. Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min. Treat cells with antibodies for α-actinin eg.  alpha sarcomeric actin antibody [alpha Sr-1] (ab28052)  and ANP diluted in the blocking buffer as above for 1 hour. Wash x3 in PBS for 5 min each. Add secondary ab in the same blocking buffer (for green ab: CY2 or fluoroscein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min (though these will require some optimization) Wash in PBS x3 Add DAPI solution to coverslips for 15 min. Wash x2 in PBS. Add mounting solution and coverslip. Image cells within 2 days. The α-actinin stain is stable for quite some time but th

Introduction to apoptosis - A brief overview of the mechanisms of apoptosis

Apoptosis or programmed cell death is defined as " a mechanism of cellular suicide which occurs after sufficient cellular damage ". Apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs and eventually these blebs separate from the dying cell and form "apoptotic bodies". Apoptotic cells also cease to maintain phospholipid asymmetry in the cell membrane, and phosphotidylserine appears on the outer leaflet. The mitochondrial outer membrane also undergoes changes that include loss of its electrochemical gradient, and substances like cytochrome c leak into the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic bodies and the dying cell. The apoptotic cell does not provoke an inflammatory response, and only individual cells are affectedby apoptosis in vivo . The mechanisms of apoptosis Apoptosis can be induced in response to

DNA fragmentation analysis protocol - Semi-quantitative method for measuring apoptosis by Richard Pattern (Tufts-New England Medical Center)

Procedure: Pellet cells Lyse cells in 0.5 ml detergent buffer: 10 mm Tris (pH 7.4), 5 mm EDTA, 0.2% Triton  Vortex  Incubate on ice for 30 min. Centrifuge at 27000 g for 30 minutes  Divide supernatants into 2-250 μl aliquots  Add 50 ul ice cold 5 M NaCl to each followed by vortexing  Precipitate DNA: Add 0.6 ml 100% EtOH and 150 μl 3 M Na acetate, pH 5.2 place at –80oC freezer for 1 hr. Centrifuge 20,000 g for 20 min and pool DNA extracts by re-dissolving by adding 400 μl total) of extraction buffer (10 mM Tris and 5 mm EDTA).  Add 2 μl (10 mg/ml) DNase free RNase. Incubate for 5 hr at 37 o C.  Add 25 μl Proteinase K at 20 mg/ml and 40 μl of buffer (100 mm Tris, pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65 o C.  Extract DNA with phenol, choloform, isoamyl alcohol and precipitate with EtOH. From Kotamraju et al JBC, 2000 DNA Fragmentation: A distinctive feature of apoptosis at the biochemical level is DNA fragmentation. This method was used as a semiquantitati

Caspase detection protocol Procedure for detection and immunostaining of caspases, including list of available kits

Introduction Caspases are essential in cells for apoptosis, one of the main types of programmed cell death in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Caspases were first implicated in apoptosis when CED-3, a protein required for programmed cell death in Caenorhabditis elegans, was found to have close homology with the mammalian interleukin-1 -converting enzyme (ICE or caspase 1) and that over-expression of ICE induced apoptosis. Failure of apoptosis is one of the main causes of tumour development and autoimmune diseases. This coupled with the unwanted apoptosis that occurs with ischaemia or Alzheimer's disease, has raised interest in caspases as potential therapeutic targets. Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. As proteases, they are enzymes that cleave other proteins. They are called cysteine proteases, because they use a cyste

Apoptosis induction protocols - Procedure for biological and chemical induction of apoptosis in cells

Apoptosis may be induced in experimental systems through a variety of methods. In general, they can be divided into 2 categories: a) biological induction; and b) chemical induction. A) Biological induction of apoptosis Activation of either Fas or TNF-receptors by the respective ligands or by cross-linking with agonist antibody induces apoptosis of Fas- or TNF receptor-bearing cells. Below is general protocol used to induce apoptosis using anti-Fas receptor (CD95) mAb in Jurkat Cells. 1. Grow Jurkat cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified, 5% CO 2 incubator at 37°C. 2. Suspend the cells in fresh medium at a concentration of 1 × 105 cells/ml. After two to three days of incubation in a 37°C, 5% CO 2 incubator, harvest the cells by centrifugation at 300–350 × g for 5 mins. 3. Resuspend cells in fresh medium to 5 × 105 cells/ml and add CD95 mAb to a final concentration of 0.05 – 0.1 μg/ml. Incubate for 3–6 hours in a 37°C incubator. As a negative co

Annexin V detection protocol:Procedure for the early detection of apoptosis using annexin V, including list of available kits

The appearance of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell is an early parameter of apoptosis, which can also be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and therefore can be used as a probe for detecting apoptosis. Example protocol -  Protocol for Annexin V-FITC Apoptosis Detection Kit (ab14085) : A) Incubation of cells with Annexin V-FITC Induce apoptosis by desired method. Collect 1-5 x 10 5 cells by centrifugation. Resuspend cells in 500 μl of 1X Binding Buffer. Add 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, optional.) Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis. B) Quantification by flow cytometry Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) usin

Advances in Cytochemical Methods for Detection of Apoptosis

Image
Abstract In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with thi