Isolation of Mature Adipocytes from White Adipose Tissue

Day 1 afternoon

Day 2 morning

Day 2 afternoon

Day 3 morning

Day 3 afternoon

Day 4 morning Thaw “control” or normotensive strain samples 1–4 and “experimental” or hypertensive strain samples 13–16. Apply the hybridization cocktails to microarrays and hybridize overnight

Prepare solutions for washing and staining. Process “control” or normotensive strain samples 1–4 and “experimental” or hypertensive strain samples 13–16 with the fluidics station. Scan these eight arrays

Thaw “control” or normotensive samples 5–8 and “experimental” or hypertensive strain samples 17–20. Apply the hybridization cocktails to microarrays and hybridize overnight

Prepare solutions for washing and staining. Process “control” or normotensive strain samples 5–8 and “experimental” or hypertensive strain samples 17–20 with the fluidics station. Scan these eight arrays

Thaw “control” or normotensive strain samples 9–12 and “experimental” or hypertensive strain samples 21–24. Apply the hybridization cocktails to microarrays and hybridize overnight

Prepare solutions for washing and staining. Process controls 9–12 and experimentals 21–24 with the fluidics station. Scan these eight arrays to complete the generation of experimental data

2.14 Materials

2.14.1 Hybridization Components, Stock Solutions and Buffers Affymetrix manuals are available online and are very user-friendly. The protocols listed by Affymetrix should be strictly followed (http://www.affymetrix.com/support/technical/manuals.affx).

All solutions should be prepared using ultrapure water (purified deionized water with a sensitivity of 18 MW at 25 °C), unless otherwise specified, and analytical grade reagents. Prepare all stock solutions at room temperature and store at the proper temperatures listed.

1. Water, Molecular Biology Grade (Fisher Scientific, Pittsburgh,

PA). 2. Acetylated Bovine Serum Albumin (BSA) solution (50 mg/ mL) (catalog number 15561-020) (Life Technologies). 3. Herring Sperm DNA (catalog number D1811) (Promega

Corporation). 4. GeneChip® Hybridization Control Kit (catalog number 900454) (Affymetrix, Santa Clara, CA) Contains the 20×

Eukaryotic Hybridization Control Stock composed of premixed biotin-labeled bioB, bioC, bioD and cre, in staggered amounts, which is added directly in the preparation of the hybridization cocktail. These controls allow for monitoring of the hybridization process for troubleshooting. The kit also contains the Control Oligonucleotide B2 (3 nM) which is used for alignment of array probe cell features for image analysis.

2.14.2 Washing and Staining Components, Stock Solutions and Buffers 5. 5 M NaCl, RNase-free, DNase-free (Life Technologies, Grand

Island, NY). 6. MES hydrate SigmaUltra (catalog number M5287) (Sigma-Aldrich). 7. MES Sodium Salt (catalog number M5057 or M3058) (Sigma-Aldrich). 8. EDTA Disodium Salt, 0.5 M solution (catalog number E7889) (Sigma-Aldrich). 9. Dimethyl sulfoxide (DMSO) (catalog number D5879) (Sigma-Aldrich). 10. Sufact-Amps 20 (Tween-20), 10 % (catalog number 28320) (Pierce Chemical). 11. GeneChip® Rat Genome 230 2.0 Arrays (Affymetrix). 12. 12× MES stock solution: 1.22 M MES, 0.89 M [Na+].

Molecular Biology Grade water should be used in creating this solution. Add about 500 mL Molecular Biology Grade water to a 1-L graduated cylinder. Weigh 64.61 g MES hydrate and transfer to the cylinder. Weigh 193.3 g MES Sodium Salt and transfer to the cylinder. Mix and adjust the volume to 1000 mL with Molecular Biology Grade water. (A magnetic stirring bar helps to dissolve the materials into solution.) The pH should be between 6.5 to 6.7 (see Note 2). Filter the solution through a 0.2 mm filter. The solution should be shielded from light and stored at 2–8 °C. (see Note 3). 13. 2× Hybridization buffer: 100 mM MES, 1 M [Na+], 20 mM

EDTA, 0.01 % Tween-20. In a 100 mL beaker, combine 8.3 mL of 12× MES stock solution, 17.7 mL of 5 M NaCl (RNase-free, DNase-free), 4.0 mL of 0.5 M EDTA, 0.1 mL of 10 % Tween-20 and 19.9 mL ultrapure water. Mix and filter the 50 mL of solution through a 0.2 mm filter. The solution should be shielded from light and stored at 2–8 °C.

1. Streptavidin, R-Phycoerythrin Conjugate (SAPE), 1 mg/mL (catalog number S-866) (Life Technologies). 2. PBS, pH 7.2 (catalog number 20012-027) (Life Technologies). 3. UltrapureTM 20× SSPE (3 M NaCl, 0.2 M NaH2PO4, 0.02 M

EDTA) (catalog number 15591043) (Life Technologies). 4. IgG from goat serum, reagent grade (catalog number I525610MG) (Sigma-Aldrich). For 10 mg/mL Goat IgG stock, resuspend 10 mg in 1 mL of PBS, pH 7.2 (or 150 mM NaCl) and store at 4 °C. Larger volume stocks can be stored at −20 °C until use. 5. Biotinylated anti-streptavidin antibody from goat (catalog number BA-0500) (Vector Laboratories).

3 Methods

3.1 Eukaryotic Target Hybridization

6. Stringent wash buffer: 100 mM MES, 0.1 M [Na+], 0.01 %

Tween-20. In a 1-L graduated cylinder, combine 83.3 mL of 12× MES stock solution, 5.2 mL of 5 M NaCl (RNase-free,

DNase-free), 1.0 mL of 10 % Tween-20 and 910.5 mL ultrapure water. Mix and filter the solution through a 0.2 mm filter.

The solution should be shielded from light and stored at 2–8 °C. 7. Non-stringent wash buffer: 6× SSPE, 0.01 % Tween-20. In a 1-L graduated cylinder, combine 300 mL of 20× SSPE, 1.0 mL of 10 % Tween-20 and 699 mL ultrapure water. Mix and filter the solution through a 0.2 mm filter. The solution can be stored at room temperature. 8. 2× Stain buffer: 100 mM MES, 1 M [Na+], 0.05 % Tween-20.

In a 500 mL graduated cylinder, combine 41.7 mL 12× MES stock solution, 92.5 mL 5 M NaCl (RNase-free, DNase-free), 2.5 mL 10 % Tween-20 and 113.3 mL of ultrapure water. Mix and filter the solution through a 0.2 mm filter. The solution should be shielded from light and stored at 2–8 °C.

The methods presented in this manuscript are based on our experience conducting experiments using the Affymetrix GeneChip® Rat Genome 230 2.0 Arrays. The preparation of the hybridization cocktails is for use with the Affymetrix GeneChip® Rat Genome 230 2.0 Arrays, which are the standard (49 Format/64 Format) arrays (see Note 4) from Affymetrix. The samples were processed according to the Affymetrix GeneChip® Expression Analysis Technical Manual (catalog number 702232, Revision 3) (http:// media.affymetrix.com/support/downloads/manuals/expression_ analysis_manual.pdf).

Use sterile, RNase-free microcentrifuge vials and sterile barrier pipette tips for all procedures. 1. In a 1.5 mL microcentrifuge vial, mix the following to create the hybridization cocktail for each sample: 15 μg of prepared fragmented and labeled cRNA, 5 μL control oligonucleotide

B2 (3 nM), 15 μL 20× eukaryotic hybridization controls (see

Note 5), 3 μL herring sperm DNA (10 mg/mL), 3 μL acetylated bovine serum albumin (BSA) solution (50 mg/mL), 150 μL 2× hybridization buffer, 30 μL DMSO. Bring the final volume to 300 μL with nuclease-free water. 2. Allow the arrays to equilibrate to room temperature immediately before use (see Note 6). 3. Heat the hybridization cocktail to 99 °C for 5 min in a heat block (see Note 7).

3.2 Microarray Washing and Staining

4. While the sample is heating, the microarray cartridge needs to be filled with 200 μL of 1× hybridization buffer (see Note 8).

On the back of the GeneChip® cartridge, there are two rubber septa. To fill the array, first insert a clean, unused pipette tip into the upper septa for venting. Using a micropipetter, insert the tip into the remaining septa to fill with the 1× hybridization buffer for pre-hybridization wetting. Remove all tips and incubate the array filled with 1× hybridization buffer in the

GeneChip® Hybridization Oven at 45 °C for 10 min with rotation at 60 rpm. 5. Transfer the hybridization cocktail (that has been heated to 99 °C) to a 45 °C heat block for 5 min. 6. Following the 5 min incubation, spin the hybridization cocktail in a microcentrifuge for 5 min to collect any insoluble material from the hybridization mixture. 7. Remove the array from the hybridization oven. Vent the array, as above when loading, and then extract the 1× hybridization buffer. Leave the venting pipette tip in place and fill the

GeneChip® with 200 μL of the hybridization cocktail. Be sure to avoid any insoluble matter at the bottom of the microcentrifuge tube. 8. Place the sample-filled array in the hybridization oven. Rotate at 60 rpm for 16 h at 45 °C. 9. During the latter part of the overnight 16 h incubation, proceed to the following section to prepare reagents required at the end of the hybridization.

The washing and staining of the Affymetrix GeneChip® Rat Genome 230 2.0 Arrays are automated using the Affymetrix GeneChip® Fluidics Station 450. To wash, stain, and scan an array, a sample file must be created using the GCOS software (or the updated AGCC software http://www.affymetrix.com/esearch/ search.jsp?pd=131429&N=4294967292). Registering the sample file (EXP file in GCOS, ARR file in AGCC) is the beginning of the Affymetrix data flow. The created sample file will be referred to for the washing, staining, and subsequent scanning of the array by the automated instrument protocols. Samples should be processed according to the Affymetrix GeneChip® Expression, Wash, Stain and Scan User Manual (catalog number 702731, Revision 3) (http://media.affymetrix.com/support/downloads/manuals/ wash_stain_scan_cartridge_arrays_manual.pdf).

The manual provides step-by-step directions for using the Affymetrix GeneChip® Fluidics Station 450. Once samples are registered, they can be automatically processed using the manufacturer’s protocol. The manual lists materials for staining solutions based on an individual array. It is recommended that the researcher mix up the

solutions for the number of samples being processed that day and aliquot them appropriately. It is highly recommended that the user prepare for one additional solution “set” to the number being processed. If the user is processing six arrays, enough of both, the Streptavidin, R-Phycoerythrin (SAPE) solution mix and the antibody solution should be created to allow for processing of seven arrays as seen in the following example. It is important to make fresh solutions on the day of the washing and staining of the array. 1. Mix the following for the SAPE solution mix (Streptavidin,

R-Phycoerythrin stain):

Component Volume/reaction # of samples Volume for 7 reactions

2× Stain buffer 600.0 μL 7

BSA (50 mg/mL) 48.0 μL 7

Streptavidin Phycoerythrin (SAPE) 12.0 μL 7

DI H2O 540.0 μL 7

Total 1200.0 μL 7 4200.0 μL

336.0 μL

84.0 μL

3780.0 μL

8400.0 μL

2. Aliquot 600.0 μL of the SAPE solution mix into 1.5 mL microcentrifuge tubes. For the seven samples example, the user would have fourteen microcentrifuge tubes each containing 600.0 μL volume of SAPE solution mix.

Component Volume/reaction # of samples Volume for 7 reactions

2× Stain Buffer 300.0 μL 7

BSA (50 mg/mL) 24.0 μL 7

Goat Ig G stock (10 mg/mL) 6.0 μL 7

Biotinylated antibody (0.5 mg/mL) 3.6 μL 7

DI H2O 266.4 μL 7

Total 1200 μL 7 2100.0 μL

168.0 μL

42.0 μL

25.2 μL

1864.8 μL

4200.0 μL

Aliquot 600.0 μL of the antibody solution mix into 1.5 mL microcentrifuge tubes. For the samples example, the user would have seven microcentrifuge tubes each containing 600.0 μL volume of antibody solution mix. 3. Once the above solutions have been freshly prepared, remove the arrays from the hybridization oven. 4. Using venting, similar to when filling the arrays, remove the hybridization cocktail and place it in a clean microcentrifuge tube. (The hybridization cocktails can be stored at −80 °C and can be reused up to three times on other arrays.) Fill the array