Envision hrp

All of the IHC assays were performed following the manufacturer's instructions. Briefly, consecutive paraffin sections of tissue samples were prepared and incubated overnight at 4°C with primary antibodies against ADAMTS6 (dilution 1:100; Elabscience Biotechnology Co., Ltd., Wuhan, China). Then the sections were incubated with peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (EnVision/HRP, Dako, Denmark). Normal breast tissues, obtained from Cancer Molecular Diagnostics Laboratory of West China Hospital, Sichuan University, served as controls. Senior pathologists were responsible for interpreting and scoring the IHC results. The staining index was scored according to staining intensity (0, no staining; 1, weak, light yellow; 2, moderate, yellow brown; 3, strong, brown) and the proportion of positive cells (0 = 0%; 1 = < 10%; 2 = < 50%; 3 = < 75%; 4 = > 76%). An ‘immunoreactive score’ was determined to be the product of the intensity and percentage of positive cells, which ranged from 0 to 12. Cases with scores of 0–3 were defined as negative (−); 4–7 as weak (+); 8–9 as moderate (++); and 10–12 as strong (+++). Cases with scores of 0–7 were defined as the low expression group and those with scores of 8–12 were the high expression group.

Consecutive sections of 110 formalin-fixed, paraffin- embedded tumors from the same CRC patients as being used in the real-time PCR measurement were subjected to immunohistochemical (IHC) analysis for the ZC3H12A protein. Primary antibody, rabbit polyclonal ZC3H12A (Abcam; ab197976; 1:100 dilution), was used 30 minutes at room temperature. DAKO EnVision™+/HRP was used 30 minutes at room temperature, followed by DAB staining 5 minutes. The staining intensity of ZC3H12A was graded on a scale of 0 to 3 (0 for no staining, 1 for light yellow, and 3 for brown). The total score was calculated by the staining intensity multiplying the percentage of cells with immunoreactivity. The staining results were scored by two pathologists blinded to the clinical data.

Immunocytochemistry was performed on charged slide cytocentrifuge (41 ×
g for 6 minutes) preparations of BALF leukocytes from a
horse in exacerbation of SEA. Primary antibodies are listed in Table 1.
Cytocentrifuge preparations were fixed with cold acetone, incubated with primary
antibodies for 30 minutes, washed, and incubated with horseradish
peroxidase–labeled secondary anti-IgG antibody (EnVision HRP, Dako Cytomation)
for 30 minutes. Preparations were washed and counterstained with hematoxylin,
and bound antibodies were detected with NovaRED chromogen (Vector Laboratories).
Stained samples were examined by light microscopy to determine antibody
specificity.

Lungs were fixed in 4% formalin for 24 h. After fixation, samples were dehydrated and embedded in paraffin. Sections 4-µm thick were stained with hematoxylin and eosin for microscopy examination and consecutive sections were used for immunohistochemical labeling. Sections were incubated with anti-ADAMTS12 (H-142, Santa Cruz Biotechnologies, 1 h at 37 °C; 1:50 dilution) or with anti-Ki-67 (ab66155, Abcam, o/n at 4 °C; 1:4000 dilution) primary antibodies. Sections were then incubated 30 min with EnVision™+/HRP (Dako) and 5 min with Liquid DAB (Dako). Samples were counterstained with hematoxilin.

Immunohistochemistry (IHC) stain was performed using a two-step EnVision/HRP technique (Dako Cytomation, Denmark) according to the manufacturer's instruction. Polyclonal rabbit anti GR antibody (sc-8992) (dilution 1∶100) was procured from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The omission of primary antibodies was used as the negative control. Three slides were read at 10 fields of high power (400×) and cytoplasm stained with brown was scored as positive. The expression of GR was quantitatively evaluated using an Olympus BH2 microscope with computer- aided images analysis system (Qiu Wei Inc, Shanghai, China). The digital images were archived by a digital camera (Nikon 4500, Tokyo, Japan). The positive area and optical density (OD) of GR-positive cells were determined by measuring three randomly selected microscopic fields (25×10) for each slide. The IHC index was defined as average integral optical density (AIOD) (AIOD  =  positive area × OD/total area).