Cold-induced activation of brown adipose tissue and adipose ... - Nature

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Cold-induced activation of brown adipose tissue and adipose angiogenesis in mice Sharon Lim1,3, Jennifer Honek1,3, Yuan Xue1, Takahiro Seki1, Ziquan Cao2, Patrik Andersson1, Xiaojuan Yang1, Kayoko Hosaka1 & Yihai Cao1,2 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden. 2Department of Medicine and Health Sciences, Linköping University, Linköping, Sweden. 3These authors contributed equally to this work. Correspondence should be addressed to Y.C. ([email protected]). 1

© 2012 Nature America, Inc. All rights reserved.

Published online 1 March 2012; doi:10.1038/nprot.2012.013

Exposure of humans and rodents to cold activates thermogenic activity in brown adipose tissue (BAT). This protocol describes a mouse model to study the activation of BAT and angiogenesis in adipose tissues by cold acclimation. After a 1-week exposure to 4 °C, adult C57BL/6 mice show an obvious transition from subcutaneous white adipose tissue (WAT) into brown-like adipose tissue (BRITE). The BRITE phenotype persists after continuous cold exposure, and by the end of week 5 BRITE contains a high number of uncoupling protein-1–positive mitochondria, a characteristic feature of BAT. During the transition from WAT into BRITE, the vascular density is markedly increased owing to the activation of angiogenesis. In BAT, cold exposure stimulates thermogenesis by increasing the mitochondrial content and metabolic rate. BAT and the increased metabolic rate result in a lean phenotype. This protocol provides an outstanding opportunity to study the molecular mechanisms that control adipose mass.

INTRODUCTION In contrast to most other tissues in the body, adipose tissue constantly experiences expansion and shrinkage throughout adulthood because of changes in metabolic demand. The plasticity of WAT and BAT determines whether an individual phenotype is obese or lean. Obesity and its related metabolic disorders such as diabetes, cardiovascular disease and cancer are among the leading causes of mortality in adult humans in Western society and in most other parts of the world1,2. Therefore, the prevention and treatment of obesity has become a priority for improving public health, entailing joint efforts from scientific research, pharmaceutical companies and various governmental organizations. Despite tremendous efforts in adipogenesis research, an effective approach for pharmaceutical intervention for obesity is lacking. At present, the most effective nonpharmaceutical methods for combating obesity are restriction of food intake, prevention of nutrient absorption and increasing physical activities. In addition to the plasticity of adipocyte sizes, both WAT and BAT undergo marked functional alterations under various physiological and pathological conditions. For example, cancer cachexia can activate metabolic pathways in WAT adipocytes, leading to adipose atrophy3. Under physiological conditions, the exposure of rodents such as experimental mice to cold augments uncoupling protein-1 (UCP-1)-dependent nonshivering thermogenic (NST) pathways in subcutaneous WAT (sWAT) (a BRITE phenotype) via activation of the sympathetic system4–7. During this transition, angiogenesis is simultaneously activated, resulting in increased vascular density associated with increased levels of oxygen consumption2,5. Adipose tissue is also considered to be the largest endocrine organ in the body and it produces numerous growth factors, hormones, cytokines and adipokines, which act on a number of nonadipose cell types1,2,8. Thus, structural and functional alterations of the adipose tissue might have a broad impact on multiple systems in the body. Here we describe a mouse model to study cold-induced structural and functional changes in both WAT and BAT. This model permits kinetic study of the transition of sWAT adipocytes toward a BAT-like phenotype (a BRITE phenotype) in association with the activation of angiogenesis. The structural and functional changes of adipocytes and microvascular endothelial cells can be easily detected using 606 | VOL.7 NO.3 | 2012 | nature protocols

specific molecular markers for each cell type. For example, BAT and BRITE cells specifically express UCP-1 as a marker to define these cells, and endothelial cells are detected by CD31 or other specific markers. We and others have studied the transition from WAT into a BAT-like phenotype by using mouse genetic models9–11. However, those genetic models are usually based on the overexpression or deletion of a specific gene in mice and are thus less relevant to human studies. In addition, genetic manipulations of a specific pathway in adipocytes in mice may lead to alterations of gene expression or function associated with that signaling system, and thus they do not reflect a common signaling system controlling the transition. Recent studies show that a substantial amount of BAT exists in adult humans and that exposure of adult humans to cold can also activate metabolism in BAT12–15. Thus, our cold-induced BAT model in mice is clinically relevant to human subjects. As angiogenesis is required for tissue mass expansion and metabolism, the coldinduced angiogenic switch in the adipose tissue offers an excellent opportunity to study molecular mechanisms underlying microvessel growth and functions. This mouse model also provides an opportunity to study the therapeutic options of combinations of cold and potential drugs that might synergistically reduce body weight. Advantages and limitations Key advantages: • The procedure is simple and the cost is low. • Mice with different genetic backgrounds and most of the genetically modified strains, including transgenic and knockout mice, can be studied under this condition. • During cold exposure, administration of drugs, chemical compounds or protein molecules can be achieved, allowing investigators to study their impact on adipose and vascular systems. • The cold-induced BRITE and vascular phenotypes become obvious after only 1 week of exposure to 4 °C and persist for the long term as long as mice are kept in the cold environment. The turnover time for experiments is short. • There is little variation between mouse individuals in each experimental setting.

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Experimental design Cold acclimation. Adult male or female mice should be divided into two groups for 30 °C and 4 °C acclimation. To obtain statistically meaningful values, a sufficient number of mice should be enrolled in each group. Typically, no less than ten mice should be included in each group. Several extra mice in the 4 °C group should be included if you are using genetically modified mice, including various transgenic and knockout strains, owing to unpredictable responses to the cold acclimation that might lead to death of the mice. However, if the measurement of metabolic rates is planned, at least 14–20 mice should be used in each group. This is especially crucial if not only basal but also norepinephrine (NE)-induced NST is to be measured. It is important that the strain, age and sex of the mice are identical in both groups and that a single mouse is placed in each cage. We advise placing several food pellets inside the cages so that mice have easy access to food. Before exposure at 4 °C, mice should be kept at 18 °C for at least 1 week for adaptation. Otherwise, direct transfer of mice from room temperature (RT, usually 22 °C) to 4 °C could lead to a high death rate. A second group of mice can be directly transferred from RT to the 30 °C thermoneutral temperature. Both the 4 °C and 30 °C groups of mice should be exposed for an equal time period to obtain comparable experimental data. For BAT activation and WAT-BRITE conversion, mice typically need to be exposed to 4 °C for 1 week or 4–5 weeks. However, prolonged exposure to cold may further increase phenotypic changes. For gene expression analysis, we recommend that several early and late time points be considered as the expression of genes is differentially regulated over time. For the detection of adipose microvasculatures, an obvious angiogenic phenotype can be readily detected after a 1-week cold exposure; this becomes more robust after 4–5 weeks of exposure. Metabolic measurement. At the end point of the experiment, some of the mice should be considered for measurement of metabolic parameters. The remaining mice should be used for gene expression

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Limitations: • Setup of cold (4 °C) and warm (30 °C) facilities in an animal housing facility is required. • Obtaining ethical permissions for experimentation can be time consuming. • In some genetically modified mouse strains, the animals die before developing full–size adipose depots. Thus, it is difficult to perform experiments in those mice.

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Figure 1 | Postmortem dissection of iWAT and iBAT depots and low temperature–induced activation in BAT. (a) Incision steps 1–5 to locate the iWAT. Numbers indicate the sequence of incision and arrowheads indicate the direction of incision. Arrows point to iWAT depots. (b) Incision steps 1–5 to locate the iBAT. Numbers indicate the sequence of incision and arrowheads indicate the direction of incision. Arrows point to iBAT depots. (c) After a 4-week exposure to 4 °C or 30 °C, C57BL/6 mice were euthanized and adipose depots were dissected. H&E staining of iWAT and iBAT revealed the existence of a high density of intracellular organelles and smaller adipocyte sizes in 4 °C–exposed iWAT and iBAT relative to the corresponding 30 °C–exposed adipose tissues. (d) Quantitative measurement of adipocyte sizes of 4 °C– or 30 °C–exposed iWAT and iBAT. All animal studies were approved by the animal care and use committee of the Northern Stockholm Experimental Animal Ethical Committee. n = 8 samples per group. Error bars indicate s.e.m.

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profile analysis, because injection of NE into mice for metabolic measurement can affect gene expression. To measure the basal meta bolic rate, mice are simply placed in a metabolic chamber in which O2 consumption, body temperature and release of CO2 are continuously recorded for 24 h. To measure NST-related metabolism, the members of a different group of mice are anesthetized by intraperitoneal injection with pentobarbital; they are then transferred immediately into a metabolic chamber. The basal metabolic rate of each mouse should be recorded for about 30 min, followed by subcutaneous injection with NE. The NE-stimulated NST-related metabolism should be recorded for ~1 h. It is important to use different mice for basal and NE-induced metabolic measurements because the temperature settings used for standard basal metabolic measurement might otherwise affect NE response. Tissue sample collection. At the end point of experiments, mice can be euthanized by exposure to a lethal dose of CO2. In the case of collection of blood samples, intracardiac puncture should be immediately performed to collect ~800 µl of fresh blood from each mouse. As plasma and serum are prepared, nonheparin-, heparin- or other anticoagulant-coated tubes should be used. Various adipose depots can be carefully collected by surgical excision. The dissected adipose tissues from different groups of mice should be photographed to highlight the color differences. In addition, adipose depots should be weighed. It is important to include appropriate control samples from the same depot in each experimental group. After photographing and weighing the tissues, each adipose depot should be equally divided into three portions. One portion of the tissue should be immediately frozen in liquid nitrogen. The other portion should be transferred to a cryomold and covered with Tissue-Tek solution; these embedded tissues should be placed on dry ice and stored at − 80 °C until use. The third portion of the tissue should be fixed at 4 °C with 4% (wt/vol) paraformaldehyde (PFA) for 24 h and then transferred to a new tube containing a sufficient volume of 1× PBS. If needed, the fixed tissues can be further divided into two portions for whole-mount staining/regular immunohistochemistry analysis or for paraffin embedding. nature protocols | VOL.7 NO.3 | 2012 | 607

protocol Histology. Paraffin-embedded samples are suitable for H&E staining, which can reveal the morphology and size of adipocytes (Fig. 1). In addition, H&E staining also detects intracellular fine organelles such as mitochondria. Paraffin-embedded samples can also be used for immunohistochemical staining, including detection of blood vessels, mitochondrial contents and other cell types such as inflammatory cells. To obtain specific staining and high-quality positive signals,

it is imperative to choose highly specific antibodies for immunohistological staining. Notably, some antibodies may only work on paraffin-embedded samples, whereas other antibodies can only be used for frozen sections. We recommend that a skillful researcher try different conditions before performing a large-scale experiment. For whole-mount immunostaining, relatively fresh tissue samples should be used for staining, and confocal microscopy should be available.


MATERIALS REAGENTS • Mice ! CAUTION All animal studies should conform to all relevant ethics regulations and must be reviewed and approved by governmental and institutional animal care and use committees.  CRITICAL It is important to note that some genetic mouse models are sensitive to cold exposure, which may lead to a high mortality rate. • Acetone (Sigma-Aldrich, cat. no. 32201) • Alexa Fluor 555 goat anti-rat IgG (H + L) antibody (Invitrogen, cat. no. A-21434) • Antigen unmasking solution, citric acid based (Vector Laboratories, cat. no. H-3300) • Cryomold intermediate (Sakura Tissue-Tek, cat. no. 4566) • Cy5 goat anti-rabbit IgG (H + L) antibody (Invitrogen, cat. no. A-10523) • DAPI (dilactate; Invitrogen, cat. no. D3571) • Distilled water (dH2O) • Dried fat-free milk (e.g., Semper) • Dry ice • Eosin Y solution (Sigma, cat. no. 318906) • Ethanol (99.7%; Solveco AB, cat. no. 200-578-6) • Hematoxylin solution, Harris modified (Sigma-Aldrich, cat. no. HHS16) • Methanol (Sigma-Aldrich, cat. no. 32213) ! CAUTION Methanol is toxic when swallowed or when fumes are inhaled. Methanol should be kept in a chemical fume hood. Wear suitable protective clothing while handling it. • Nail polish • Nonimmune goat serum (Vector Laboratories, cat. no. S-1000) • OCT compound Tissue-Tek (Sakura Tissue-Tek, cat. no. 4583) • PBS (1×) • PFA (Sigma-Aldrich, cat. no. 441244) ! CAUTION It is hazardous when exposed to skin, inhaled or swallowed. Preparation of 4% (wt/vol) PFA should be performed in a chemical fume hood while wearing appropriate protective clothing. • Pentobarbital (Sigma-Aldrich, cat. no. P3393) • Pertex (Histolab Products, cat. no. 00801) • Proteinase K (Invitrogen, cat. no. 25530-049) • Rabbit anti-mouse prohibitin polyclonal (Abcam, cat. no. ab28172) • Rat anti-mouse CD31 monoclonal antibody (MEC13.3; BD Pharmingen, cat. no. 553370) • Triton X-100 Polyoxyethylene(10) octylphenyl ether (Acros Organics, cat. no. 215680010) ! CAUTION Harmful if swallowed. Handling should be performed in a chemical fume hood with appropriate protective clothing. • Trizma base (Sigma, cat. no. T4661-100G) • Vectashield mounting medium (Vector Laboratories, cat. no. H-1000) • Xylen (Histolab Products, cat. no. 02080)

EQUIPMENT • Room maintained at 4 °C in an animal facility suitable for keeping mice • Room maintained at 30 °C in an animal facility suitable for keeping mice • Adobe Photoshop CS3 or later versions (Adobe) • BD Falcon 50-ml polypropylene conical tubes (BD Biosciences, cat. no. 358206) • Confocal microscope (e.g., Nikon D-eclipse C1, Nikon) • Confocal software (e.g., EZ-C1 3.9 Nikon digital eclipse, Nikon) • Costar six-well cell culture plates (Corning, cat. no. 3516) • Costar 96-well cell culture plates (Corning, cat. no. 3596) • Dry-line oven (VWR, cat. no. DL53) • Forceps (AgnTho’s, cat. no. 08-060-120) • Indirect calorimeter (INCA, Somedic) • Microscope coverslips (VWR International, cat. no. 631-0135) • Microtome cryostat (Histolab Products AB, cat. no. HM500OM) • Microtome paraffin (Cellab, cat. no. Microm HM315) • Needle (BD Microlance, cat. no. 300800) • PAP pen for immunostaining (Sigma-Aldrich, cat. no. Z672548) • Plasma collection tubes (BD Microtainer, cat. no. 365975) • Rocking board (VWR International, cat. no. 444-0341) • Scalpel blade (AgnTho’s, cat. no. 02-040-010) • Scalpel blade holder (AgnTho’s, cat. no. 02-030-030) • Serum collection tubes (BD Microtainer, cat. no. 365968) • Spatula/microspoon (VWR International, cat. no. 231-1354) • Superfrost Plus microscope slides (Thermo Scientific, cat. no. 4951plus) • Syringe (BD Plastipak, cat. no. 300186) • Timer (e.g., Fisher Scientific, cat. no. FB70232)) • Vertical staining jar with glass lid (Electron Microscopy Sciences, cat. no. 70318-04) • Water bath up to 60 °C (e.g., Lauda Aqualine Al5) • MRI instrument • Scissors REAGENT SETUP PFA (4%, wt/vol) To a final volume of 500 ml, add 20 g of PFA powder to 1× PBS. At 4 °C, 4% (wt/vol) PFA can be stored for up to 2 weeks. PBST (PBS with Triton X-100 (0.3%, vol/vol)) For a final volume of 30 ml (0.3% (vol/vol) Triton X-100), add 0.9 ml of pure Triton X-100 to 29.10 ml of 1× PBS. Vortex and store the solution at 4 °C for several months. Blocking buffer (3%, wt/vol) Add 3 g of dried fat-free milk powder to 100 ml PBST to prepare 100 ml of 3% (wt/vol) blocking buffer. Blocking buffer should be freshly prepared for each experiment.

PROCEDURE Cold-induced activation of adipose tissue ● TIMING 2–8 weeks 1| Adaptation of mice (day 1). Divide the mice into at least two groups, depending on the experimental demand. Each group should consist of at least six to eight mice, sufficient for statistical analysis. Adapt one group of mice at 18 °C for 1–3 weeks and another group at RT (22 °C). ! CAUTION All animal studies must be reviewed and approved by relevant governmental and institutional animal care and use committees. A pilot experiment with a limited number of mice should be performed if a genetically modified mouse strain is used. In particular, the deletion of overexpression of metabolically related genes may cause a high rate of mortality. 608 | VOL.7 NO.3 | 2012 | nature protocols

protocol  CRITICAL STEP The adaptation time varies among different mouse strains. For wild-type C57BL/6 mice, for example, acclimation of 1 week is sufficient. Some strains, such as Ucp1–/– mice, need to be adapted at 18 °C for 3 weeks. Shorter adaptation time might cause death of mice during subsequent cold exposure. The following protocol is suitable for wild-type mice; adjust if you are using mice with special requirements. During acclimation at 18 °C, mice should preferably be kept in single cages to prevent them from being in close proximity to each other and keeping each other warm. This could otherwise interfere with proper activation of NST.  CRITICAL STEP To perform late–time point analysis, keep mice at 30 °C and 4 °C, respectively, for 4–5 weeks. 2| Day 7. Transfer the mice acclimated at 22 °C to 30 °C and mice acclimated at 18 °C to 4 °C. At the thermoneutral temperature (30 °C), mice have their lowest metabolic activity as the surrounding temperature allows the mice to maintain their optimal body temperature.  CRITICAL STEP Each individual mouse should be kept in a single cage. Check the condition of the mice daily in order to detect potential discomfort to the animals as a result of cold exposure.


3| Maintain the mice at 30 °C and 4 °C, respectively, for 1–5 weeks. ? TROUBLESHOOTING 4| Measure the body composition of the mice by MRI at the end of 30 °C and 4 °C exposure. Put the mice back in their cages after the measurement. 5| If you wish to measure the basal metabolic rate and oxygen consumption by using an indirect calorimeter, follow option A. If you wish to measure NE-stimulated NST-related metabolism, follow option B. If you wish to proceed directly to tissue extraction for gene array analysis, proceed with option C to euthanize the mice and collect tissues.  CRITICAL STEP Measurement of basal metabolic rate, oxygen consumption and NE-stimulated NST-related metabolism can affect gene expression of tissues, and therefore tissues should be collected immediately if gene array analysis is required. (A) Measurement of basal metabolic rate and oxygen consumption with an indirect calorimeter ● TIMING 1 d (i) Prepare indirect calorimeters and equip each with a timer regulating a 12-h-light to 12-h-dark cycle according to the circadian rhythm of the mice. (ii) Open the calibration gases (15% and 20% oxygen) and air supply. (iii) Switch on the cooling/temperature regulating system and metabolic chamber. (iv) Ensure that the appropriate settings function optimally (temperature and duration of measurement) for the measurement on the computer connected to the indirect calorimeters. (v) Start calibration of the metabolic chamber. Calibration is finished when a stable baseline is obtained, typically within 20–30 min. (vi) During calibration, prepare a single cage for each mouse. Equip the cage with sufficient chow and water. Measure the body weight and other parameters of interest, such as the amount of food provided for each mouse. (vii) When a stable calibration baseline is obtained and the desired temperature (e.g., 23 °C at an oxygen concentration of 21%) has been reached in the chamber, close the calibration gases. (viii) Place one mouse in the prepared cage and put the cage in the metabolic chamber. (ix) Close the chamber and continue the measurement. (x) End the measurement after 24 h and euthanize the mice as described in option C.  CRITICAL STEP To assure reproducibility of results, we recommend measuring for 24 h, as there are differences in the metabolic rate between the light and dark cycles. (B) Measurement of NE-stimulated NST-related metabolism ● TIMING 1 d (i) Open the calibration gases (15% and 20% oxygen) and air supply. (ii) Switch on the cooling/temperature regulating system and indirect calorimeters. (iii) Ensure that the appropriate settings function optimally (temperature and duration of measurement) for the measurement on the computer connected to the indirect calorimeters. Commonly, a temperature of 33 °C is used to measure NE-induced NST-related metabolism. (iv) Start the calibration of the metabolic chamber. The calibration is finished when a stable baseline is obtained, typically after 20–30 min. (v) Anesthetize the mice with pentobarbital (40–60 mg kg − 1) by intraperitoneal injection. Ensure that the mice are fully asleep.  CRITICAL STEP It is very important to choose the correct dose of pentobarbital. The effective window is very narrow and too low a dose might result in premature awakening, whereas too high a dose can be lethal for mice. ? TROUBLESHOOTING nature protocols | VOL.7 NO.3 | 2012 | 609


protocol (vi) Transfer each mouse into a single metabolic chamber. (vii) Measure the basic metabolic rate for 30 min without interruption. (viii) Open the metabolic chamber and inject NE (1 mg kg − 1) into each mouse. Subcutaneous dorsal injection is recommended. (ix) Close the metabolic chamber and continue the measurement.  CRITICAL STEP Avoid unnecessarily opening the metabolic chamber during the measurement in order to prevent disturbing the sleeping mice. (x) Measure the metabolic rate until the NE response decreases or until the mice start to wake up (typically around 1 h after NE injection). (xi) Proceed with option C to euthanize the mice and collect the tissues.  CRITICAL STEP Note that after the measurement of NE-stimulated NST-related metabolism, gene expression levels can be markedly altered. We therefore do not recommend using mice that have undergone NE stimulation for gene expression analysis. (C) Necropsy and localization of adipose depots ● TIMING variable; depends on the number of mice (i) Euthanize each mouse by a lethal dose of CO2 narcosis, followed by cervical dislocation. (ii) Depending on your experimental needs, collect blood samples by intracardial puncture. If the collection of blood samples is not necessary, proceed to Step 2C(iii). To collect blood, position each mouse with the abdomen facing up and make an incision with a pair of scissors at a 30° angle from the abdomen toward the neck. Carefully cut through the rib cage, puncture the heart with a 23-G syringe (1 ml) and gently remove blood with the syringe. Transfer the blood to plasma or serum collection tubes depending on your experimental designs. It is important that a sufficient amount of blood (usually no less than 500 µl per mouse) be collected from each mouse in order to perform multiple analyses. (iii) Localization and dissection of inguinal WAT (iWAT). Position each mouse with the abdomen facing up (Fig. 1a, left) and make an incision with a pair of scissors at a 30° angle on the ventral side from the neck to the lower abdomen (Fig. 1a, left, Step 1). Lift up the skin carefully and make four incisions toward the front and hind limbs (Fig. 1a, left, Steps 2–5). Carefully open up the cut skin and secure with needles. Dissect the iWAT depot carefully (Fig. 1a, right). ? TROUBLESHOOTING (iv) Localization and dissection of interscapular BAT (iBAT). Position each mouse with the dorsal side facing up (Fig. 1b, left) and make an incision with a pair of scissors at a 30° angle from the dorsal back toward the neck (Fig. 1b, left, Step 1). Lift up the skin carefully and make four incisions toward the front and hind limbs (Fig. 1b, left, Steps 2–5). Carefully open up the cut skin and dissect the iBAT (Fig. 1b, right). ? TROUBLESHOOTING (v) If a whole mount of adipose tissue (Step 6, option A) or H&E staining of paraffin-embedded adipose tissues (Step 6, option B) is desired, transfer the adipose depots into freshly prepared 4% (wt/vol) PFA and keep the tissues in 4% (wt/vol) PFA at 4 °C for 24 h. If immunohistochemical staining of cryosections (Step 6, option C) is required, place the adipose tissues into a plastic cryomold and add OCT compound Tissue-Tek for embedding. Collection and fixation of adipose tissues 6| If desired, perform either a whole mount of adipose tissue (option A), H&E staining of paraffin-embedded adipose tissues (option B) or immunohistochemistry on adipose cryosections (option C). (A) Adipose tissue whole mount ● TIMING 4 d (i) Preparation of adipose tissue samples (day 1). Transfer and immerse 4% (wt/vol) PFA-fixed mouse adipose tissues in Petri dishes filled with 1× PBS. The volume of the PBS should be sufficient to cover the tissues.  CRITICAL STEP To obtain optimal results, adipose tissues should always be obtained as fresh samples shortly after dissection and be immersed in the working solution (PBS or PBST). ? TROUBLESHOOTING (ii) To prepare thin slices (typically 5 mm × 5 mm), secure the adipose tissues with forceps and section them using a scalpel blade. The cuts should be even in order to produce sections of equal thickness throughout the entire tissue sample. Therefore, apply slight pressure while cutting the tissue with the scalpel blade. ? TROUBLESHOOTING (iii) Transfer the adipose tissue into a properly labeled six-well plate and wash it with ~1.5 ml of 1× PBS per well at RT for 1 h on a rocking board (adipose tissue should be covered completely with PBS). In this step, the remaining PFA is removed from the tissue. (iv) Incubate the tissue sections with Proteinase K (20 µg ml − 1 in 10 mM Tris-HCl buffer (pH 7.4)) at RT for 5 min to digest the tissue. (v) Permeabilize the adipose tissues with 100% methanol at RT for 30 min. Because of methanol toxicity, this step should be performed in a chemical fume hood. (vi) Wash the adipose tissues three times with 1× PBS for 1 h on a rocking board. 610 | VOL.7 NO.3 | 2012 | nature protocols


protocol (vii) Incubate adipose tissue with 3% (wt/vol) blocking buffer for 12–24 h at 4 °C on a rocking board to block unspecific binding sites. (viii) Primary antibody staining (day 2). Transfer the tissue into PBST and wash it thoroughly to remove blocking buffer. Then incubate the tissue sections with one or several primary antibodies. Perform antibody incubation for 12–24 h at 4 °C on a rocking board. If you are using the monoclonal rat anti-mouse CD31 antibody as the primary antibody, dilute it 1:200 in PBST. Add a sufficient volume of antibody solution to immerse the entire tissue. (ix) Secondary antibody staining (day 3). Wash the tissue samples with PBST for 1.5 h at 4 °C on a rocking board. (x) Immerse the tissue samples with 3% (wt/vol) blocking buffer for 1.5 h at 4 °C on a rocking board. (xi) Prepare dilutions of secondary antibodies in 3% blocking buffer and incubate the adipose tissues with the diluted antibody solutions for 2 h at RT on a rocking board. Use, for example, an Alexa Fluor 555-conjugated goat anti-rat antibody (dilute 1:400) as the secondary antibody. (xii) Incubate the adipose tissue sections with blocking buffer (1:1 dilution with PBST) for 1 h at RT on a rocking board. (xiii) Wash the adipose tissues at 4 °C with PBST overnight on a rocking board. (xiv) Tissue mounting (day 4). Transfer the stained tissue samples onto microscope glass slides and mount with one or two drops of Vectashield mounting medium per slide. Cover the tissue with microscope coverslips. Examine the slides directly under confocal microscopy or store the slides at 4 °C in the dark; use within a few days.  PAUSE POINT Seal the coverslips with nail polish to prevent tissues from drying; slides can be stored for up to 4 weeks at − 20 °C. ? TROUBLESHOOTING (xv) Imaging. Capture 3D images of whole-mount stained adipose tissues with a confocal microscope using a Nikon D-eclipse C1 and EZ-C1 3.90 software (or, alternatively, any equivalent confocal microscope and imaging system). Obtain images at ×10, ×20, ×40 or ×60 magnifications of 5 µm thickness. Scan eight to ten layers (choose the thickness and the number of layers according to the desired information that should be extracted from the images). Collect the images and analyze them in a quantitative manner with Adobe Photoshop. ? TROUBLESHOOTING (B) H&E staining on paraffin-embedded adipose depots ● TIMING 1 d (i) Tissue section preparation (day 1). Prepare sections of paraffin-embedded adipose tissue samples using a microtome (thickness: 5 µm for iWAT, 3 µm for iBAT). Transfer the sections with a forceps into a water bath (~40 °C). Collect the paraffin sections using a Superfrost glass slide after the paraffin surrounding the tissue has smoothed out. Air-dry the sections before proceeding to the next step. ? TROUBLESHOOTING (ii) Put the adipose tissue slides in an oven and incubate them for 2 h at 60 °C to remove excess paraffin. Allow the slides to cool to RT.  PAUSE POINT The slides can be kept for 1–2 years at RT in a storage box. (iii) Deparaffinization. Transfer the slides to a vertical staining jar. Deparaffinize the adipose tissue slides in Xylen twice (5 min each).  CRITICAL STEP Perform Step 6B(iii–xiii) by using a vertical staining jar. (iv) Rehydration of the tissue slides. Rehydrate the adipose tissue slides with 99.7% (vol/vol) ethanol twice (5 min each). (v) Rehydrate the adipose tissue slides with 95% (vol/vol) ethanol twice (5 min each). (vi) Rehydrate the adipose tissue slides with 70% (vol/vol) ethanol twice (5 min each). (vii) Wash the adipose tissue slides with dH2O for 5 min. (viii) Stain the adipose tissue slides with hematoxylin for 3 min. (ix) Remove excess hematoxylin under running water for 10 min. (x) Stain the adipose tissue slides with eosin for 1–2 min. (xi) Dehydrate the adipose tissue slides with 95% (vol/vol) ethanol twice (5 min each). (xii) Dehydrate the adipose tissue slides with 99.7% (vol/vol) ethanol twice (5 min each). (xiii) Remove the adipose tissue slides from the vertical staining jar and allow the slides to dry on the bench. (xiv) Mounting. Add one or two drops of Pertex (depending on the size of the tissue) onto the stained adipose tissue sections and cover them with microscope coverslips very gently at an angle; this will help to prevent trapping of air bubbles.  PAUSE POINT H&E-stained adipose tissue slides can be stored in a storage box for several years. (xv) Imaging. Analyze the stained adipose tissues with a bright-field microscope. Capture images at ×10, ×20, ×40 or ×60 magnifications, depending on the desired information about tissue structures. Quantify the adipocyte size using Adobe Photoshop or alternative free software (ImageJ from NIH, available at http://rsbweb.nih.gov/ij/).

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protocol (C) Immunohistochemistry on adipose cryosections ● TIMING 3 d (i) Tissue embedding (day 1). Place the cryomold containing OCT compound on dry ice to quickly freeze the sample. Upon freezing, the OCT compound solidifies.  PAUSE POINT Samples can be stored in − 80 °C for 2 years. (ii) Tissue sectioning. Before sectioning, adjust the microtome cryostat to − 30 °C. Note that the time required to achieve a temperature decrease depends on the equipment.  CRITICAL STEP For cutting of adipose tissues, the sectioning temperature is − 10 °C lower than for other types of tissues. This choice of temperature is necessary to ensure the integrity of tissue structures. ? TROUBLESHOOTING (iii) Prepare tissue sections of 15 µm thickness per slide. Carefully place the tissue samples on Superfrost Plus microscope slides.  CRITICAL STEP Compared with other nonadipose tissues, sections prepared from adipose tissues are thicker; this ensures the integrity of tissue slides.  PAUSE POINT The slides can be stored at − 80 °C for 2 years. (iv) Tissue staining (day 2). Adapt the tissue slides at RT for ~30 min. (v) In a vertical staining jar, fix the tissue with 100% acetone for 10 min. (vi) Wash the slides at least three times in 1× PBS (5 min each). (vii) Use a PAP hydrophobic pen to encircle the tissue samples for immunostaining. Encircling the tissues helps to minimize the amount of reagent required. Without touching the sections, carefully remove excess water from the slides by using a piece of paper towel (this is also applied in subsequent steps that require addition of goat serum or antibodies). (viii) To block nonspecific antigen-binding sites, incubate slides with 1× PBS containing 4% (vol/vol) normal goat serum. During this process, the slides should be positioned horizontally in a humidified incubation chamber. (ix) Wash the slides at least three times in 1× PBS (5 min each). (x) Dilute one or several primary antibodies to the desired concentrations in PBS containing 4% (vol/vol) goat serum and incubate the tissue slides with the antibody solution overnight at 4 °C in the humidified incubation chamber. For example, use rabbit anti-mouse Prohibitin as the primary antibody at a 1:100 dilution. (xi) Tissue staining (day 3). Wash the slides at least three times in 1× PBS (5 min each). (xii) Prepare appropriate dilutions in PBS containing 4% (vol/vol) goat serum for the corresponding secondary antibodies. For example, use goat anti-rabbit Cy5 at a 1:400 solution. Incubate the slides with the secondary antibody solution for 45 min at RT. Protect the slides from light. (xiii) Wash the slides at least three times in 1× PBS (5 min each). (xiv) Use a paper towel to remove the excess PBS. To mount the slides, add one or two drops of Vectashield mounting medium onto the tissues and carefully cover the tissue sections with microscope coverslips.  PAUSE POINT Mounted slides can be kept at 4 °C in the dark if they will be examined within a few days. The slides can be stored for several weeks at − 20 °C. (xv) Imaging. Analyze stained adipose tissues with a fluorescence microscope. Positive signals can be acquired by using a fluorescent microscope equipped with different excitation wavelengths. (xvi) Take images at ×10, ×20, ×40 or ×60 magnifications, depending on the desired information about tissue structures. Quantify the adipocyte size with Adobe Photoshop. ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. Table 1 | Troubleshooting table. Step

Problem

Possible reason

Solution

3

High death rate of mice in 4 °C group

Mice were adapted in groups at 18 °C

Increase the number of mice in each group; adapt mice in individual cages at 18 °C

Too short a period of adaptation at 18 °C

Prolong 18 °C adaptation

Genetic defects in some mouse strains

Optimize the adaptation condition and increase the number of animals; change the genetic background (continued)

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protocol


Table 1 | Troubleshooting table (continued). Step

Problem

Possible reason

Solution

5B(v)

Death of mice after pentobarbital injection

Amount of pentobarbital injection was too high

Weigh each mouse carefully and calculate accurate amount of pentobarbital required; also consider that certain strains might require a different dosage of pentobarbital

5C(iii), 5C(iv)

It is difficult to localize different adipose depots in transgenic mice (e.g., FOXC2 and ob/ob); lack of sufficient adipose depots

Genetically modified strains of mice and wild-type mice are phenotypically different in adipose depots

Carefully separate adipose depots from other depots by skillful dissection

Pathological conditions such as cachexia

Correct the pathological condition before cold experiments

6A(i)

Loss of adipose tissues

Accidental discarding of adipose tissues during exchanging of buffers

Pour adipose tissues into Petri dishes during exchange of buffer instead of discarding the solution directly into the sink

6A(ii)

Adipose tissues are difficult to cut into even and thin sections for whole mount

Blunt scalpel blade

Replace new blades or scissors frequently

Incorrect position of the scalpel blade

Position the scalpel blade perpendicularly to adipose tissue

6A(xiv)

Autofluorescence of stained adipose tissue

Drying out of adipose tissue because Add sufficient Vectashield mounting medium of insufficient Vectashield mounting to cover adipose tissue before covering it medium gently with coverslips

6A(xv)

Nonspecific fluorescent background

4% (wt/vol) PFA is not fresh

Use fresh 4% (wt/vol) PFA

Insufficient blockade of nonspecific binding sites

Increase incubation time with 3% (wt/vol) blocking buffer

6B(i)

Damaged paraffin-embedded adipose tissue sections

Blunt cryotome blade

Replace with a new blade frequently

6C(ii)

Cryomold-embedded adipose tissue sections are difficult to cut

Blunt cryotome blade

Replace with a new blade before problem arises

● TIMING Steps 1–5, cold-induced transformation of adipose tissue: 2–8 weeks Step 5A, measurement of metabolic rate and oxygen consumption using an indirect calorimeter: 1 d Step 5B, measurement of NE-stimulated NST-related metabolism: 1 d Step 5C, euthanizing mice and postmortem localization of adipose depots: variable; depends on the number of mice, typically 20–30 min per mouse Step 6A(i–xiv), adipose tissue whole mount: 4 d Step 6A(xv), imaging: variable; depends on the number of samples and availability of microscopes Step 6B(i–xiv), H&E staining on paraffin-embedded adipose depots: 1 d Step 6B(xv), imaging: variable; depends on the number of samples and availability of microscopes Step 6C(i–iii), preparation of tissue sections: 1 d Step 6C(iv–xiv), staining of cryosections: 3 d Step 6C(xv, xvi), imaging: variable; depends on the number of samples and availability of microscopes


protocol a

30 °C

b

4 °C

P < 0.01 8

0

iWAT

Figure 2 | Increase of prohibitin-positive mitochondria in adipocytes by cold exposure. (a) After 4 weeks of exposure to 4 °C or to 30 °C, C57BL/6 mice were euthanized and adipose depots were dissected. Cryosections of iWAT and iBAT were stained with a rabbit anti-mouse prohibitin polyclonal antibody (red) and DAPI (blue). Both 4 °C–exposed iWAT and iBAT showed an increased density of prohibitin mitochondria (red) as compared with their 30 °C counterparts. (b) Quantification of prohibitin-positive structures of 4 °C– or 30 °C–exposed iWAT and iBAT. All animal studies were approved by the animal care and use committee of the Northern Stockholm Experimental Animal Ethical Committee. n = 8 samples per group. Error bars indicate s.e.m.

iBAT

ANTICIPATED RESULTS As cold-induced BAT activation and the WAT-BAT transition are highly reproducible in each individual mouse, the variation among individual mice should be minimal. It is important that adipose tissue from the same depot be used for comparative studies. Exposure of C57BL/6 mice to 4 °C for 4 weeks generates highly reproducible examples of activation of BAT (Fig. 1c,d). In the 4 °C–exposed iWAT, the average size of adipocytes is significantly smaller relative to the 30 °C–exposed group. In addition, adipocytes in the 4 °C–exposed iWAT contained a high density of intracellular organelles (Fig. 1c,d), which are confirmed to be mitochondria expressing prohibitin (Fig. 2). Unlike iWAT, the average adipocyte size of iBAT is not signi ficantly reduced as compared with the 30 °C group (Fig. 1c,d). However, the intracellular number of mitochondria of BAT adipocytes is markedly increased at 4 °C (Figs. 1 and 2). CD31 staining of adipose microvessels demonstrated that the vascular density was markedly increased in both 4 °C–exposed iWAT and iBAT relative to their corresponding 30 °C–exposed depots (Fig. 3a,b). These results represent an example of the cold-induced transition from sWAT into a BRITE phenotype and the switch to an angiogenic phenotype. More detailed experimental findings are described elsewhere5,10. As expected, cold exposure significantly increased the capacity of NST in response to NE stimulation (Fig. 3c). The data presented in Figure 3c were adopted and modified from our previous publication (ref. 5).

Note: Supplementary information is available via the HTML version of this article. Acknowledgments We thank B. Cannon and J. Nedergaard at the Stockholm University for providing the animal facility for our research. The author’s laboratory was supported by research grants from the Swedish Research Council, the Swedish Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute distinguished professor award, the Torsten Soderbergs Foundation, the European Union Integrated Project of Metoxia (project no. 222741) and the European Research Council advanced grant ANGIOFAT (project no. 250021). AUTHOR CONTRIBUTIONS Y.C. designed the study. S.L., J.H., Y.X. and T.S. performed the experiments. S.L., J.H., Y.X., T.S. and Y.C. analyzed the data. S.L., J.H. and Y.C. wrote the paper. Z.C., P.A., X.Y. and K.H. participated in developing these protocols. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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Vo2 (ml min–1 kg–1)

4 °C

30 °C

4 °C

30 °C

CD31+ vessels area per field (×103 µm2)

iWAT

4 °C Figure 3 | Increase of vascular density and a 30 °C b c nonshivering thermogenesis in response to cold exposure. (a) After 4 weeks of exposure to 4 °C 10 P < 0.01 120 or to 30 °C, C57BL/6 mice were euthanized and 4 °C 100 8 30 °C adipose depots were dissected. Fresh iWAT and P < 0.01 80 6 iBAT were stained with a rat anti-mouse CD31 60 NE monoclonal antibody. Both 4 °C–exposed iWAT 40 4 and iBAT showed an increased vascular density 20 2 as compared with their 30 °C counterparts. 0 20 40 60 (b) Quantification of CD31-positive microvessels 0 50 µm iWAT iBAT Time (min) of 4 °C– or 30 °C–exposed iWAT and iBAT. (c) Metabolic rates of 4 °C– or 30 °C–exposed groups were measured for assessment of nonshivering thermogenesis in response to NE. The data presented in c were adopted and modified from our previous publication5. All animal studies were approved by the animal care and use committee of the Northern Stockholm Experimental Animal Ethical Committee. n = 8 samples from 4 mice in each group. Error bars indicate s.e.m. iBAT


50 µm

4 °C

P < 0.01

2

30 °C

4

30 °C

6

4 °C

iBAT

Prohibitin+ mitochondria per field (× 103 µm2)

iWAT

10

80

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