Ho1

Fig. 2. LPS induces HO-1 protein in heart and lung tissues. Total heart and lung protein was extracted from mice treated with (+) or without (-) LPS for 24 hr. Aliquots (25 /xg) were subjected to Western blotting with polyclonal anti-HO-1 antiserum.

before incubation with horseradish peroxidase-conjugated goat anti-rabbit serum (Amersham, Piscataway, NJ), diluted 1:4000, for 1 hr at room temperature. The blots are washed with 4% (w/v) milk in TBST two times (15 min each), and then with TBST two times (10 min each). The blots are then processed with an enhanced chemiluminescence reagent (Pierce) and exposed to X-ray film. Include fluorescent tape on the edge of the gel to determine the position of the molecular weight standards. An example of an HO-1 Western blot analysis of protein extracts isolated from LPS-treated mice is shown in Fig. 2.

Heme Oxygenase Enzyme Activity Assay

Two methods are routinely used to measure HO enzyme activity: (1) measurement of bilirubin generation by spectrophotometry20,21 and (2) determination of CO production by gas chromatography.22-24 The easier access to a spectrophotometer compared with gas chromatography equipment renders the colorimetric assay the method of choice for many investigators. Neither assay distinguishes the relative contribution of each HO isoform to total enzyme activity.

For the spectrophotometric determination of HO enzyme activity in the tissue and cultured cell extracts, use the following protocol.25'26

Preparation of Microsomal Extracts

1. Harvest tissues as described under Preparation of Total Protein from Mouse Tissues (above). Place the tissues (3 ml/g tissue) in ice-cold homogenization buffer

20 M. D. Maines, N. G. Ibrahim, and A. Kappas, J. Biol. Chem. 252,5900 (1977).

21 M. D. Maines and A. Kappas, J. Biol. Chem. 253,2321 (1978).

22 H. J. Vreman and D. K. Stevenson, Anal. Biochem. 168, 31 (1988).

23 M. N. Cook, K. Nakatsu, G. S. Marks, B. E. McLaughlin, H. J. Vreman, D. K. Stevenson, and J. F. Brien, Can. J. Physiol. Pharmacol. 73, 515 (1995).

24 D. M. Suttner and P. A. Dennery, FASEB J. 13,1800 (1999).

25 S.-F. Yet, A. Pellacani, C. Patterson, L. Tan, S. C. Folta, L. Foster, W.-S. Lee, C.-M. Hsieh, and M. A. Perrella, J. Biol. Chem. 272,4295 (1997).

26 A. Pellacani, P. Wiesel, A. Sharma, L. C. Foster, G. S. Huggins, S.-F. Yet, and M. A. Perrella, Circ. Res. 83, 396 (1998).

[30 mM Tris-HCl, 0.25 M sucrose, 0.15 M NaCl (pH 7.5)] containing Complete protease inhibitor (Roche) (other protease inhibitor cocktails maybe substituted, from example, Sigma protease inhibitor). For cultured cells, wash the cells with cold PBS twice, and scrape with 1.5-3 ml of homogenization buffer per 150-mm dish. Homogenize the tissues or cells with a Polytron on ice as described under Preparation of Total Protein from Mouse Tissues (above). Filter the homogenate through two layers of cheesecloth to remove large debris if present.

2. Centrifuge the homogenates at 10,000g for 15 min at 4°.

3. Collect the supernatant and centrifuge at 100,000g for 1 hr at 4°.

4. Resuspend the microsomal pellet in 50 mM potassium phosphate buffer (pH 7.4) containing protease inhibitor and sonicate. Determine the protein concentration by the BCA method as described under Western Blot Analysis of Heme Oxygenase 1 (above).

Rat Liver Microsomal Supernatant as Source ofBiliverdin Reductase

1. Perfuse rat liver through the portal vein with cold PBS to flush out blood. Take 3 g (~2 small lobes) of liver and wash twice in ice-cold PBS.

2. Transfer the liver tissue to a small beaker containing 10 ml of ice-cold homogenization buffer (plus protease inhibitors); mince the tissue with scissors.

3. Transfer the minced tissue to a 50-ml tube and homogenize on ice with a Polytron equipped with a 12-mm probe.

4. Filter the homogenate through two layers of cheesecloth to remove large debris.

5. Centrifuge at 10,000g for 15 min at 4°. Filter the supernatant through two layers of cheesecloth. Repeat the spinning.

6. Collect the supernatant and centrifuge at 100,000g for 1 hr at 4°. The microsomal supernatant fraction is used as a source of biliverdin reductase for the HO enzyme assay. Aliquot the supernatant and store at -80° until use.

Heme Oxygenase Activity Assay

1. All reactions should be prepared on ice, using precooled reagents.

2. Prepare 500-/zl reactions in duplicate in amber 1.5-ml microcentrifuge tubes as follows. Prepare a blank reaction for each sample by replacing the NADPH-generating system with an equivalent volume of 0.1 M phosphate buffer, pH 7.4.

Microsomal fraction x ¡x\

Potassium phosphate buffer (pH 7.4), 50 m M 90-* ¡il

Potassium phosphate buffer (pH 7.4), OA M 177 ¡x\

NADPH-generating system (see note 1) 167 /u.1

(substitute with 167 fi\ of 0.1 M phosphate buffer, pH 7.4, for blank) Hemin solution, 2.5 mM (see note 2) 6.7 ill

Add hemin solution last and vortex immediately; return the tubes to ice. Note 1: Assemble the NADPH-generating system by mixing equal volumes of each of the following components:

Note 2: Prepare hemin (Sigma) solution in the dark immediately before use: to 1.63 mg of hemin add 0.2 ml of 0.2 N NaOH, vortex to dissolve, and then add 0.8 ml of 0.1 M potassium phosphate buffer, pH 7.4.

3. Incubate the reactions for 10-20 min at 37° in the dark.

4. Terminate the reaction by placing the samples on ice for at least 2 min.

5. The absorbance spectrum of each reaction is scanned between 450 and 550 nm, using a prechilled quartz cuvette with a spectrophotometer (Beckman, Fullerton, CA).

6. Calculate the difference in optical density between 530 nm and the peak (~462-464 nm) for each sample. The amount of bilirubin formed is determined as AOD464-530 nm (extinction coefficient, 40 mlf1 cm-1 for bilirubin).

7. To calculate the total HO enzyme activity, subtract the bilirubin concentration in each sample reaction from the corresponding blank reaction. HO activity is expressed as nanomoles of bilirubin formed per milligram of protein per hour.

Lipid Peroxidation Measurements

A method to assess oxidative damage in tissues is to measure lipid peroxidation products from tissue extracts. Mouse tissues are harvested and washed in ice-cold 20 mMTris, pH 7.4, blotted dry, and weighed. Homogenize the tissue in 20 mM Tris, pH 7.4 (10%, w/v), using a Polytron as described above. Centrifuge the homogenate at 3000g for 10 min at 4°. Transfer the supernatant to new tubes. The colorimetric lipid peroxidation assay kit (Calbiochem, San Diego, CA) is used to measure malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) levels in the supernatant from LPS-treated mouse tissues13 according to the manufacturer instructions.

1. Prepare diluted reagent 1 (Rl): 3 parts R1 to 1 part methanol.

2. To 650 jLtl of diluted Rl in a 1,5-ml polypropylene microcentrifuge tube add 200 fil of sample (brought to a final volume of 200 pA with 20 mM Tris,

MgCl2

NADP (Sigma)

Glucose 6-phosphate (Sigma)

Glucose-6-phosphate dehydrogenase (Sigma; diluted

in 0.1 M phosphate buffer, pH 7.4) Potassium phosphate buffer, pH 7.4

20 units/ml 0.1 M

pH 7.4) or standard solution (see below). Vortex for 3-4 sec. For standards, dilute 4-HNE standard (SI) or MDA standard (S2) solutions 100-fold (v/v) in sample buffer to 100 \iM. To cover the standard concentration range of 0-20 ijM, use 0-200 fi\ of 100 ¡x,M standard solution, brought to a final volume of 200 /¿I with the same sample buffer (for samples containing low levels of MDA/4-HNE, prepare a standard concentration range of 0-5 ¡iM).

3. To assay MDA plus 4-HNE:

b. Mix well and close the caps.

d. Chill the samples on ice.

e. Spin the samples at 13,000g for 10 min to pellet cellular debris if samples are cloudy.

f. Transfer 200-/xl aliquots of supernatant to a 96-well plate.

g. Determine the OD5g6 value.

4. To assay MDA only:

a. Add 150/Ltl of 12WHC1.

b. Mix well and close the caps.

d. Chill the samples on ice.

e. Spin the samples at 13,000g for 10 min to pellet cellular debris if samples are cloudy.

f. Transfer 200-/nl aliquots of supernatant to a 96-well plate.

g. Determine the OD586 value.

5. Plot a standard curve and calculate the concentration of samples.

Histological Analysis Tissue Preparation for Histology

Tissues from mice subjected to endotoxemia, myocardial ischemia and reperfusion, or hypoxia are harvested for histological analysis to measure HO-1 expression and inflammatory markers. Before harvest, mice are anesthetized and the chest is opened. The heart is perfused with a 22-gauge catheter through the left ventricle, advancing the catheter toward the outflow of the aorta. The right atrium is nicked with scissors to permit outflow of the blood and solutions. The hearts are first perfused with PBS (5-10 ml) to flush out blood, followed by 10% (w/v) neutral buffered formalin (NBF; Sigma). Good perfusion is indicated by a uniform pale appearance of the kidneys and a contraction of the skeletal muscle with the NBF perfusion Remove the heart and other organs and continue to fix in at least 20 volumes of 10% (w/v) NBF at 4° overnight. If samples are not immediately processed as described below, transfer the tissue to PBS and store at 4° to prevent overfixation.

For certain histological stains and immunostaining, hearts are removed after perfusing with PBS only, fixed in methyl Carnoy's (MC) solution [60% (v/v) methanol, 30% (v/v) chloroform, 10% (v/v) acetic acid] at 4° on a shaker for 5 hr, and then changed to 70% (v/v) ethanol and allowed to incubate at 4° overnight. Heart samples are then processed in an automated tissue processor (Hypercenter XP; Shandon, Pittsburgh, PA) according to the following protocol: 50% (v/v) ethanol for 20 min; 70% (v/v) ethanol for 20 min; 80% (v/v) ethanol for 20 min; 95% (v/v) ethanol for 20 min; 95% (v/v) ethanol for 20 min; 100% ethanol for 20 min; 100% ethanol for 30 min; xylene for 30 min; xylene for 30 min; paraffin for 45 min; paraffin for 45 min.

Tissue is then removed from the processor and embedded in paraffin (Paraplast X-tra; Electron Microscopy Sciences, Fort Washington, PA). The specimens are sectioned on a microtome (Leica, Bannockburn, IL) at a thickness of 5 fim, using standard techniques.

Immunohistochemical Detection of Heme Oxygenase 1 Protein and Inflammatory Cell Markers

Immunohistochemistry is performed as follows to detect the expression of HO-1 protein in the heart and other tissues. An example of HO-1 expression in the heart is shown in Fig. 3.

1. Paraffin is removed from the tissue sections, using a hot air dryer (Shandon), until the paraffin around the sections has melted (do not exceed 60°).

2. The sections are deparaffinized and rehydrated with quick rinses through xylene (three times), 100% ethanol (twice), 95% (v/v) ethanol (twice), 70% (v/v) ethanol (once), 50% (v/v) ethanol (once), and distilled H2O.

Control

Fig. 3. Immunohistochemical localization of HO-1 expression in the heart after ischemia and reperfusion. Heart sections from (A) a control, nonoperated mouse and (B) a mouse subjected to 1 hr of ischemia and 24 hr of reperfusion (I/R) were stained with a polyclonal anti-HO-1 antibody. Brown staining indicates HO-1 expression. Original magnification: x400.

3. Slides can be mounted in an IHC Rack&Rinse rack (Shandon) to conserve antibodies and minimize solution volumes. Alternatively, use a PAP pen to outline (Electron Microscopy Sciences) the area surrounding the section and perform immunostaining in a humidified chamber. With either method, it is essential that sections do not dry out during the staining procedure. Incubate the sections with Cadenza buffer (Shandon; 5 min each, two times). Note: PBS or Tris-buffered saline may be substituted for Cadenza buffer.

4. Incubate the sections in 3% (v/v) H2O2 in Cadenza buffer [900 /A of Cadenza plus 100 iA of 30% (v/v) H2O2] at room temperature for 20 min.

5. Wash with Cadenza buffer (twice, 5 min each time).

6. Incubate the sections for 20 min at room temperature with 10% (v/v) normal goat serum (Vector Laboratories, Burlingame, CA) in Cadenza buffer, 200 /xl/slide.

7. Incubate the sections with polyclonal anti-HO-1 antiserum (SPA-895; StressGen Biotechnologies) diluted 1:400 in Cadenza buffer (200 ¿¿1/slide) at room temperature for 1 hr, and then at 4° overnight.

8. Rinse the slides in Cadenza buffer (twice, 5 min each time).

9. Incubate the sections in secondary antibody [biotinylated anti-rabbit IgG(H+L), made in goat; Vector Laboratories] at a 1:200 dilution at room temperature for 1 hr.

10. Rinse the slides in Cadenza buffer (twice, 5 min each time).

11. Incubate the sections with Elite Vectastain ABC HP solution (200 /¿l/slide; Vector Laboratories) at room temperature for 1 hr. This solution should be made up at least 30 min before use and keep at 4°.

12. Rinse the slides as in step 8.

13. Remove from the IHC Rack&Rinse rack a slide to be used for determining the optimal developing time. Develop the slide with a Vector 3,3'-diaminobenzidine (DAB) substrate kit by putting drops of substrate on the slide to cover the sections. Once the sections are covered by substrate, monitor the enzyme reaction closely under a microscope to see the color gradually develop. Stop the development by immersion in water as soon as signal is seen and before any nonspecific (background) staining appears. Develop the rest of the slides, which are still mounted on the IHC Rack&Rinse rack (200 /il of substrate per slide), for the same period of time.

14. Wash the slides in water for 5 min.

15. Counterstain the sections with methyl green [1% (w/v) in water] for 1015 min for nuclear staining. Wash under running water to rinse out excess staining. Slides are dehydrated through 70% (v/v) ethanol for 30 sec; 95% (v/v) ethanol for 30 sec; 95% (v/v) ethanol for 30 sec; 100% ethanol for 30 sec; 100% ethanol for 2 min; xylene for 2 min; and xylene for 2 min.

16. Mount the slides with coverslips, using Permount/xylene (50:50, v/v) and allow to air dry.

WT TG

Leukocytes

Neutrophils

Leukocytes

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