Identification of the Cellular Content of Healing Infarcts

During the proliferative phase of healing, infarcts contain a large number of inflammatory, vascular, and matrix-producing cells. To study the cellular environment in healing infarcts, we developed immunohistochemical methods to identify cell types often associated with mast cells in healing tissues. This section describes immunohistochemical techniques to label neutrophils, macrophages, endothelial cells, fibroblasts, and smooth muscle cells in normal and infarcted canine hearts.

3.2.1 Fixation and Tissue Processing

For optimal staining with the endothelial cell (CD31) and macrophage (PM-2K) markers we use samples fixed in B*5 fixative without formalin (0.6% zinc chloride) as described by Beckstead (23). This fixative combines optimal antigenic survival with good morphological preservation.

3.2.2. Immunohistochemistry

1. Immunohistochemistry is performed using the ELITE mouse kit, which contains all the necessary reagents for the technique except the hydrogen peroxide, PBS, the primary antibodies and the peroxidase substrate kit (diaminobenzidine).

2. 5-( sections are cut, dewaxed in xylene and graded alcohols, and rehydrated.

3. Sections are pretreated with a solution of 3% hydrogen peroxide to inhibit endogenous peroxidase activity and incubated with 2% horse serum for 1 h to block nonspecific protein binding.

4. Subsequently they are incubated for 2 h at room temperature with the primary antibody diluted in 2% horse serum. The following primary antibodies are used: mouse anti-human macrophage antibody PM-2K (1:200 dilution) to identify mature canine macrophages (24), mouse anti-human Mac 387 antibody (1:1000 dilution) to detect newly recruited myeloid cells (neutrophils and monocytes), mouse anticanine neutrophil antibody SG8H6 (kindly donated by Dr. C. W. Smith, Baylor

College of Medicine [25]), mouse anti-human CD31 antibody (1:100 dilution) to label endothelial cells, and mouse anti-human a-SMA antibody (1:400 dilution) to identify myofibroblasts and smooth muscle cells (see Note 4).

5. After rinsing with PBS three times, 5 min each slide, the slides are incubated for 30 min with the secondary antibody (0.5% biotinylated anti-mouse IgG) and then again washed in PBS.

6. The biotinylated secondary antibody is detected using the ABC system, a preformed macromolecular complex between avidin and biotinylated enzyme. Sections are incubated for 30 min with ABC reagent, which is prepared 30 min before incubation according to the manufacturer's recommendations.

7. After washing with PBS, peroxidase activity is detected using diaminobenzidine (Peroxidase substrate kit).

8. Slides are counterstained with eosin and mounted in Cytoseal XYL mounting medium.

9. Mast cell identification is performed with a rabbit anti-canine antibody to chymase at a 1:500 dilution (kindly donated by Dr. G. H. Caughey, UCSF [19]) using the ELITE rabbit kit. The immunohistochemical protocol is similar to the one described previously for the murine antibodies; however a biotinylated anti-rabbit IgG (included in the kit) is used as the secondary antibody.

4. Notes

1. Mast cell heterogeneity was established by the pioneering work of Enerback, who demonstrated a distinctive mucosal mast cell phenotype in the gastrointestinal tract of the rat (26,27). In rodents, the use of modified fixation helps to distinguish mast cell subpopulations: mucosal or "atypical" mast cells are smaller in size, and their granules may become resistant to metachromatic staining after routine fixation with formalin, whereas "typical" or connective tissue mast cells contain large amounts of histamine and stain metachromatically regardless of fixation. In humans, the strict classification into mucosal and connective tissue type mast cells is not possible. However, human mast cell subtypes can also be defined according to staining and fixation properties (28). We have shown that the canine heart contains at least two subpopulations of mast cells, which can be distinguished by their staining properties: approx 40% of the mast cells stained with toluidine blue when the tissue was fixed with formalin (resembling "typical" or "connective tissue" mast cells), whereas the remainder could not be detected unless fixatives such as Carnoy's were used (2).

2. Fluorochrome-conjugated avidin is a simple and reliable technique for detection of mast cells in the canine heart. Avidin binds specifically to individual mast cell granules allowing identification of degranulated mast cells in areas of injury (29). TRITC-avidin and FITC-avidin are equally effective in identifying mast cells. Bergstresser and colleagues suggested that fixation is unimportant for mast cell staining with FITC-avidin (21). However, in our experience, infarcted formalin-fixed canine hearts demonstrated fewer mast cells compared with samples fixed in Carnoy's. The effects of different fixatives on mast cell staining with conjugated avidin have not been systematically studied.

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Fig. 3. Identification of the cellular content of canine infarcts (A and B, 1-h occlu-sion/24-h reperfusion; C-F, 1-h occlusion/7-d reperfusion). (A) After 24 h of reperfusion staining with Mac387, an antibody that labels newly recruited myeloid cells (monocytes and neutrophils) identifies a large number of leukocytes in the myocardium (arrowhead). (B) In comparison, at the same time point, staining with PM-2K, a marker for mature macrophages labels a small number of cells (arrowheads). (C) After 7 d of reperfusion the healing infarct is filled with PM-2K positive mature macrophages. (D) Mast cells are identified using immunohistochemistry for chymase.

(E) CD31 immunohistochemistry labels the vascular endothelium in the healing infarct.

(F) a-SMA staining identifies myofibroblasts, predominantly localized in the infarct border zone (arrowheads) and vascular smooth muscle cells (arrow).

Fig. 3. Identification of the cellular content of canine infarcts (A and B, 1-h occlu-sion/24-h reperfusion; C-F, 1-h occlusion/7-d reperfusion). (A) After 24 h of reperfusion staining with Mac387, an antibody that labels newly recruited myeloid cells (monocytes and neutrophils) identifies a large number of leukocytes in the myocardium (arrowhead). (B) In comparison, at the same time point, staining with PM-2K, a marker for mature macrophages labels a small number of cells (arrowheads). (C) After 7 d of reperfusion the healing infarct is filled with PM-2K positive mature macrophages. (D) Mast cells are identified using immunohistochemistry for chymase.

(E) CD31 immunohistochemistry labels the vascular endothelium in the healing infarct.

(F) a-SMA staining identifies myofibroblasts, predominantly localized in the infarct border zone (arrowheads) and vascular smooth muscle cells (arrow).

3. Tryptase histochemistry has been used to identify mast cells in human, bovine, canine, and murine tissues. Cytochemical reaction mixtures need to be made fresh daily. In addition, the sensitivity of the technique decreases in old histological samples. In our experience, only a small percentage of mast cells can be identified in tissues older than six months. When using fresh samples, however, the method is relatively simple, reliable, and specific for identification of canine mast cells. It is particularly valuable because commercially available antibodies to human tryptase do not crossreact with its canine homologue.

4. Identification of specific cell types in canine tissues requires use of optimal cellular markers (Fig. 3). Most anti-human CD68 antibodies do not crossreact with canine species. However, the monoclonal antibodies AM-3K and PM-2K, specific for mature human macrophages, detect canine macrophages in all tissues examined (24,30). Endothelial cell identification requires the use of an anti-CD31 antibody (31). Factor VIII staining labels only arteriolar and venular but not capillary endothelial cells. In addition, lectin histochemistry identified vascular endo-thelial cells in control hearts, but gave poor results in staining of the infarct vasculature (31). The antibody Mac387 labels myeloid cells, neutrophils and monocytes, but not mature macrophages and serves as a marker of acute inflammatory activity (30,32). In control hearts a-SMA staining labels vascular smooth muscle cells. However in healing infarcts, a large population of phenotypically modulated fibroblasts termed myofibroblasts express a-SMA and are predominantly localized in the border zone of the infarct (33).

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  • Brodie
    Why slides were incubated with biotinylated secondary antibody?
    8 years ago

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