Ultra 5 block

Representative hematoxylin and eosin-stained glass slides containing tumoral lesion specimen from the 152 CCRCC patients were reviewed and selected by the 2 pathologists. Two 3-mm cores were obtained from each representative intratumoral lesion in the paraffin block and transplanted to recipient tissue microarray (TMA) blocks. Immunohistochemical staining was performed on 4-μm sections of the TMA block samples. The sections were attached to glass slides, deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Each section was heated for 20 minutes in 10 mM citrate buffer (pH 6.0) in a microwave oven (700 W). After incubation with Ultra V block (LabVision Corporation, Fremont, CA, USA) for 7 minutes at room temperature to block background staining, the slides were incubated with an MIF monoclonal primary antibody (1:1000 dilution, ab55445, Abcam, Cambridge, United Kingdom). An ultraView Universal DAB detection kit was used (760–500, Ventana, Tucson, AZ, USA) according to the manufacturers recommendation. 3, 3′-Diaminobenzidine was used to detect the protein reactivity. The sections were counterstained using hematoxylin.

Immunohistochemistry for caspase-3 (rabbit polyclonal antibody, CPP 32, Ab-4, RB-1197-R7, ready to-use for immunohistology, Lab Vision, Fremont, CA, USA) was performed using a combination streptavidin–biotin–peroxidase method and microwave antigen retrieval on formalin-fixed paraffin-embedded tissues. After deparaffinization, sections were treated with 10% hydrogen peroxidase in filtered water to block endogenous peroxidase activity. To retrieve the antigen, slides were boiled with 10 mmol/l citrate buffer (pH 7) for 10 min. After preincubation with Ultra V block (Lab Vision) for 20 min, sections were incubated with the primary antibody for 1 hour at room temperature, followed sequentially by biotinylated goat antipolyvalen (Lab Vision) for 20 min and streptavidin peroxidase complex (Lab Vision) for 20 min. We used 3,3'-diaminobenzidine tetrahydrochloride/DAB (Lab Vision) as the chromagen, and hematoxylin for nuclear counterstain, and then rinsed and mounted. Omission of the primary antibody created the negative control, and tonsil was used as the positive control. The slides were evaluated in a blinded manner by the same investigator. Immunoreactivity was scored using a semiquantitative scale for intensity of staining as follows: 0 (negative, no staining), 1+ (weakly positive), 2+ (moderately positive), and 3+ (strongly positive).

Tissue sections of paraffin-embedded formalin-fixed tissue blocks were deparaffinized with xylene for 5 min each, followed by two washes with 100% ethanol for 10 min. The slides were then incubated in 95% ethanol for another 10 min and washed with dH₂O twice for 5 min. Antigen retrieval was performed by placing slides in 10 mmol/l citrate buffer (pH 6.0) and microwave treatment for 15 min. Tissue sections were cool down to room temperature (RT), washed in phosphate-buffered saline (PBS) and distilled water. Afterwards, sections were blocked with Ultra V Block (Lab Vision, Westinghouse Drive, Fremont, CA, USA) for 4 min. After a consecutive PBS wash, slides were incubated with the monoclonal Mouse anti cadherin-11 IgG2B Clone # 283416 Catalog Number: MAB1790 (R&D Systems). Negative controls were performed on all tissue sections by replacing primary antibodies with diluted isotype immunoglobulin (ImmunoCruz™ Staining system, Santa Cruz Biotechnology). Then the slides were incubated with goat anti-polyvalent and streptavidin-HRP (both Lab Vision) for 60 min, followed by an incubation with 3-amino-9-ethylcarbazole (AEC). Finally, slides were washed in PBS, counterstained with hematoxylin for 5 sec and cover-slipped.