Table 2 Antibodies used for immunohistochemical evaluation

CD3

T-lymphocytes

rabbit pc IgG

A0452

DAKO

EDTA buffer pH9 (Target Retrieval®, Dako)

1/100

0.5% dilution of horse biotinylated secondary antibody + ABC amplification system, Vector Laboratories

45 minutes - 95°C

CD4

helper and regulatory T-lymphocytes

1 F6

mouse mc IgG1

NCL-CD4-1 F6

NOVOCASTRA

10 mM Citrate Buffer pH6

1/20

non diluted anti-mouse Envision® system, Dako

4B12

mouse mc IgG1 κ

NCL-CD4-368

NOVOCASTRA

3 x 5 minutes – 750 W

1/20

non diluted anti-mouse Envision® system, Dako

CD8

cytotoxic T-lymphocytes

C8/144B

mouse mc IgG1κ

#M7103

DAKO

EDTA buffer pH9 (Target Retrieval®, Dako)

1/50

non diluted anti-mouse Envision® system, Dako

45 minutes - 95°C

CD20cy

mature and immature B-lymphocytes

L26

mouse mc IgG2a κ

M7055

DAKO

10 mM Citrate Buffer pH6

1/200

0.5% dilution of horse biotinylated secondary antibody + ABC amplification system, Vector Laboratories

2 minutes - 750 W + 7 minutes -250 W

CD56

Natural Killer cells

564

mouse mc IgG2b

NCL-CD56-504

NOVOCASTRA

10 mM Citrate Buffer pH6

1/50

non diluted anti-mouse Envision® system, Dako

45 minutes – 95°C

CD79αcy

B cells from early pre-B to plasma cell stage and neoplastic B-lymphocytes

HM57

mouse mc IgG1κ

M7051

DAKO

EDTA buffer pH9 (Target Retrieval®, Dako)

1/50

0.5% dilution of horse biotinylated secondary antibody + ABC amplification system, Vector Laboratories

45 minutes - 95°C

TIA-1

CD4 and CD8 positive T cells, granulocytes, monocytes and activated NK cells

2G9

mouse mc IgG1

PN IM2550

IMMUNOTECH

10 mM Citrate Buffer pH6

1/50

non diluted anti-mouse Envision® system, Dako

2 minutes - 750 W + 7 minutes -250 W

MHC Class I

most nucleated cells

HC10

mouse monoclonal IgG2a

SB03 546

kindly provided by Dr S. Ferrone, G. Gaslini Institute, Italy

EDTA buffer pH9 (Target Retrieval®, Dako)

1/20

non diluted anti-mouse Envision® system, Dako

45 minutes - 95°C.