From LipidomicsWiki

Contents 1 Equipment 2 Reagents 3 Secondary antibody-gold conjugates 4 Biological Specimens used for Lipid Droplet Studies 4.1 Human THP-1 monocyte/macrophages. 4.2 3T3-L1 cells (adipocytes). 4.3 Human milk 4.4 Mounting and freezing the specimens. 4.5 Preparing freeze-fracture replicas 4.6 SDS treatment and immunolabeling of replicas

Equipment

•  Balzers freeze-fracture machine (e.g., BAF 301) equipped with electron beam guns and quartz crystal thin film monitor.
•  Balzers gold/nickel alloy specimen carriers, flat-topped type.
•  Two liquid nitrogen bench dewars, one equipped with inner receptacle for primary coolant, and one with stainless steel specimen  holding basket.
•  Fine forceps of the type used for electron microscopy
•  Binocular microscope
•  Porcelain dishes with wells for replica processing
•  Replica transfer implement - glass Pasteur pipette with tip melted to give a fine knob
•  Sonicator
•  Copper grids (200 mesh)
•  Transmission electron microscope

Reagents

•  Cell culture media and supplements as detailed below.
•  Phosphate buffered saline (PBS)
•  Tris-HCl buffer (Tris)
•  Glycerol
•  Ethanol
•  Cryogens: liquid nitrogen; nitrogen slush, Freon or propane.
•  Sodium dodecyl sulphate (SDS) solution: 5% SDS (Serva) in 10 mM Tris, 30mM sucrose
•  Blocking solution: 5% BSA in PBS
•  0.5% EM grade glutaraldehyde in PBS
•  Primary antibodies:
•  Adipophilin: mouse monoclonal antibody raised against a peptide matching amino acids 5-27 from the amino terminus of human  adipophilin (AP 125; Progen Biotechnik, Heidelberg, Germany).
•  TIP47: guinea pig polyclonal antibody raised against peptide matching amino acids 1-16 from the amino terminus of human TIP47(GP30; Progen Biotechnik, Heidelberg, Germany).
•  Perilipin: guinea pig polyclonal antibody raised against a synthetic peptide corresponding to the N-terminal region of human perilipin A  and B (GP30; Progen Biotechnik, Heidelberg, Germany). 
  
• Xanthine oxidoreductase: rabbit polyclonal antibody raised against bovine xanthine oxidoreductase (R1119P; Acris Antibodies, Hiddenhausen, Germany)
•  Butyrophilin: polyclonal antibodies raised in rabbit against 2 sequences, one in the N-terminus and one in the C-terminus (26).
•  Caveolin-1: mouse monoclonal antibody raised against caveolin-1 (Clone 2297, BD Transduction Laboratories, Lexington, KY, USA).

Secondary antibody-gold conjugates

•  Goat anti-mouse, goat anti-rabbit and donkey anti-guinea pig coupled to 6nm, 12nm or 18nm colloidal gold (Jackson Immunoresearch,
•   West Grove, PA, USA)
  

Biological Specimens used for Lipid Droplet Studies

Human THP-1 monocyte/macrophages.

1. Culture human THP-1 monocytes (American Type Culture Collection, Manassas, VA, USA) in suspension at 37°C in a 5% CO2  atmosphere in RPMI 1640 medium with recommended supplements.
   
2. Add 100 µM phorbol 12-myristate 13-acetate to the medium for 3 days to stimulate the cells to differentiate into adherent   macrophages.
   
3. Add 50-100 µg/ml acetylated low density lipoprotein (AcLDL) at day 2 for 24 – 48 hours to induce lipid droplet formation. 

3T3-L1 cells (adipocytes).

1. Maintain 3T3-L1 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine fetal calf serum (FCS), 2 mM glutamine, 100 µg/ml penicillin and 100 µg/ml streptomycin, at °C in a 5% CO2 atmosphere.
2.  To induce differentiation of 3T3-L1 cells to adipocytes, incubate confluent cell monolayers in DMEM supplemented with 10% FCS, mM isobutylmethylxanthine, 10 µg/ml insulin, and 10 µM dexamethasone for 72 h.
3. To induce lipid droplet formation, change medium to 50 µg/ml acLDL in DMEM supplemented with 10 µg/ml insulin and 10% FCS for  48 hours.
   

Human milk

1. Gently centrifuge fresh milk (e.g., 4,000 rpm in an Eppendorf bench centrifuge model number 5415D).
2. Collect supernatant containing suspended milk fat globules (and occasional mammary epithelial cells)

Mounting and freezing the specimens.

1. Prepare equipment for freezing the specimens either by:
1. pouring liquid nitrogen into dewar flask and then dispensing primary coolant (e.g., Freon or propane) into inner receptacle; or,
2. pouring liquid nitrogen into insulated container and placing under vacuum (subjection to several cycles of evacuation may be
    necessary) to produce nitrogen slush (i.e., liquid nitrogen cooled below its boiling point).
2. Pour liquid nitrogen into a second bench dewar equipped with stainless steel specimen collecting basket
3. Place several cleaned gold/nickel alloy flat-topped specimen carriers under binocular microscope in readiness for mounting the
    samples.
4. For cell samples, scrape cells from culture dish using rubber policeman or take suspensions of cells directly, and either:
    1. Place sample in 30% glycerol in PBS, gently centrifuge to concentrate (12,000 x g, 30 seconds), and mount on the specimen
        carriers; or,
    2. Place a droplet of 30% glycerol in PBS directly on the carriers, pellet cells without initial glycerol treatment and add fragments of
        cell pellet to the glycerol on the carriers.
5. For milk samples, place the supernatant directly on the specimen carrier in a droplet of 30% glycerol in PBS.
6. Inspect mounted specimens using the binocular microscope ensuring that the sample appears as a concentrated hemispherical
    mound on the flat top of the carrier. Carefully remove excess liquid from the specimen if necessary using the tips of fine forceps.
7. If using a primary coolant (Freon or propane), melt the frozen coolant using a metal rod. If using nitrogen slush, remove the coolant
    from the vacuum. Grip the flange of the carrier using fine forceps and plunge the mounted specimen into the coolant, and then 
    immediately transfer to liquid nitrogen in second bench dewar equipped with holding basket. Repeat so that all specimens are frozen
    quickly in succession. Once the specimens have been frozen, they may be transferred for longer term storage in a liquid nitrogen
    refrigerator or processed immediately for freeze fracture.

Preparing freeze-fracture replicas

1. Set up the electron beam guns for oblique (38°) platinum/carbon evaporation (electrode 1) and vertical carbon only evaporation
    (electrode 2). Check that the microtome knife is clean and undamaged; replace if necessary.
2. Pump out vacuum chamber, test guns and thin film monitor, and switch the specimen table on to cooling.
3. When the specimen table has reached -150°C, organize frozen mounted specimens adjacent to door of freeze-fracture machine, in
    readiness for insertion.
4. Bring chamber of freeze-fracture machine to atmospheric pressure, remove any condensation on the cold table using liquid Freon, 
    rapidly insert frozen specimens onto the cold table, close the chamber door, and evacuate.
5. As the chamber is pumping out, switch on cooling of the microtome, and adjust temperature of the table to bring the specimens to -
   100°C.
6. When the knife has completed cooling (i.e., the lowest temperature attainable with the liquid nitrogen supply lines) and the vacuum has
    reached 5 x 10-6 mbar, fracture the specimens (now at -100°C) by microtome knife; take one or more sweeps with the knife while
    observing with the binocular microscope.
7. Evaporate platinum/carbon from electrode 1, immediately after the final sweep of the knife (settings: 1,800V, 70-80 mA) to deposit a
    film of average 2nm thickness determined with the quartz crystal monitor.
8. Evaporate carbon from electrode 2 (settings: 2,400V, 120-140 mA) to deposit a film of 24nm thickness determined with the quartz
    crystal monitor.
9. Bring chamber of freeze-fracture machine to atmospheric pressure, remove replicated specimens with fine forceps and place under
    PBS in a well of a porcelain spotting dish.

SDS treatment and immunolabeling of replicas

1. Place spotting dish under the binocular microscope and, using fine forceps, pluck visible cellular material from the replica.
2. Dispense 5% SDS in 10 mM Tris/30mM sucrose (pH 8.3) into a separate porcelain spotting dish. Use a glass tip to transfer the replica
    from the PBS into the SDS, with observation under the binocular microscope.
3. Gently agitate the SDS solution using a shaking machine. Cover replica and leave in SDS for 20-24 hours at room temperature. From
    time to time, place under the binocular microscope and continue tugging away any visible strands of biological material using forceps.
4. Pipette PBS into an unused well of the dish, and transfer replica into the PBS. Wash for 1 hour.
5. For immunolabeling, transfer replica into successive wells of dish at room temperature as follows: i) blocking solution (15 minutes), ii)
    primary antibody in blocking solution (1 hour), iii) PBS, three changes (total 30 minutes), iv) secondary antibody in blocking solution (1
    hour), v) PBS, three changes (total 30 minutes), vi) 0.5% glutaraldehyde in PBS (10 minutes), vii) distilled water, two changes. 
    Controls are conducted in parallel (e.g., secondary antibody alone to check for non-specific binding to the replica, irrelevant primary
    antibody etc).
6. Mount replicas on electron microscope grids, and examine in transmission electron microscope.