Polymer hrp kit

Lung tissue from five patients with culture-proven M. tuberculosis was studied. Control tissue was obtained from uninvolved lung parenchyma of patients who underwent a surgical procedure for lung cancer. MT1-MMP immunoreactivity was evaluated using the mouse mAb 114-6G6 (Abcam, ab77965). Briefly, sections were rehydrated in graded alcohols and heated in a microwave oven at 900 W for 20 min in citrate buffer at pH 6. They were cooled at room temperature before immunostaining. MT1-MMP mAb was used at a concentration of 20 μg/ml for 2 h at room temperature and then processed with a polymer-HRP kit (BioGenex, San Ramon, CA) with diaminobenzidine development and Mayer hematoxylin counterstaining. Breast and colon tissues were used as a positive external control. Negative controls were obtained by omitting the primary Ab and using competing peptide used to raise the primary Ab. Slides were reviewed with a histopathologist, and images were taken using an Olympus BX51 microscope. This project was approved by the Hammersmith and Queen Charlotte’s Research Ethics Committee (reference no. 07/H0707/120).

A tissue microarray (TMA) containing normal ovarian tissue (n = 40) and ovarian cancer cores (n = 40) was obtained from US Biomax (OV801). Other TMAs were developed in Imperial College as detailed in Results. Paraffin was removed with Histoclear and sections were rehydrated in graded alcohols and heated in a microwave oven at 900W for 15 min in citrate buffer pH 6. Tissue sections were pretreated using 0.3% H₂O₂ in phosphate bufferedsaline (PBS), rinsed in PBS and then incubated with 20 μl/ml normal goat serum. Primary antibodies, anti-LARP1 (SDIX-Novus Biologicals) or anti-Ki67 antibody (Leica) were diluted in PBS and incubated overnight at 4°C. Secondary or biotinylated-secondary antibodies were incubated for 30 min at room temperature and then processed with the Polymer-HRP Kit (BioGenex), or stained using the Vectastain Elite ABC Kit (Vector labs) and ImmPACT DAB (Vector labs). Tissues were then counterstained with haematoxylin. Intensity of staining was defined for each specimen (0–3, with 3 being most intense) multiplied by the percentage of cells stain-positive (to give a total score out of 300). All images were captured using a Nikon Eclipse ME600. Xenograft samples were processed in the same manner as clinical TMAs.