The paraffin blocks were cut at 5 μm, after which the
slides were randomly coded by an outside laboratory technician. The
code was broken only after macrophage and survival data were ready for
analysis, with all investigators masked to the outcome of individual
patients until that time. Immunostaining of macrophages was performed
using the avidin-biotinylated peroxidase complex method (Vectastain ABC
Elite Kit; Vector Laboratories, Burlingame, CA), as described
previously in detail.
18
Two primary mouse monoclonal antibodies (mAbs) PG-M1
(IgG
3; lot 101, diluted 1:50; Dakopatts,
Klostrup, Denmark) and KP1 (IgG
1; lot 038,
diluted 1:100; Dakopatts) were used to recognize fixative-resistant
epitopes on CD68, an intracytoplasmic 110-kDa glycoprotein that resides
in lysosomal granules and is expressed by macrophages in all human
tissues.
19 20 A third mAb, clone 3A5 against human
macrophages (IgG2b; lot 210501, diluted 1:50; Novocastra Laboratories,
Newcastle-upon-Tyne, UK), was also used. Pretreatment with 0.4%
(wt/vol) pepsin (FIP, 2500 U/g; E. Merck, Darmstadt, Germany) in 0.01 M
hydrochloric acid for 15 minutes at 37°C enhanced immunostaining with
mAb PG-M1, and pretreatment in 10 mM sodium citrate buffer (pH 6.0) for
10 minutes at 95°C enhanced immunostaining with mAbs KP1 and 3A5. In
preliminary experiments, mAb 3A5 immunolabeled dendritic macrophages
less effectively than mAb PG-M1, in accordance with a previous
study,
21 and mAb KP1 labeled fewer macrophages overall
than mAb PG-M1 did. mAb KP1 also cross-reacted with melanoma cells in
several tumors, as has been noted earlier in cutaneous
melanoma.
19 22 Consequently, mAb PG-M1 was used throughout
this study as the default antibody to quantitate macrophages. The
primary mAb QBEND/10 (lot 121202, diluted 1:25; Novocastra) to the CD34
epitope of endothelial cells was used to label
microvessels.
13
To enable evaluation of immunoreaction in pigmented tumors, the
peroxidase reaction was developed with 3,3′-diaminobenzidine
tetrahydrochloride and, regardless of the grade of pigmentation,
melanin was then bleached with 3.0% (vol/vol) hydrogen peroxide and
1.0% (wt/vol) disodium hydrogen phosphate, as described
previously.
18