Human breast tissue biopsy samples and tumor tissue arrays
Twenty fresh biopsy samples (9 malignant and 11 benign) from female patients were obtained from Creighton University Medical Center (CUMC), Omaha, NE (IRB #0513792). Patient identifiers were coded to protect confidentiality. The fresh tissue was flash-frozen by placing on dry ice and then cut into two sections for RNA and protein analysis. Paraffinized blocks of breast biopsy patients and their subsequent surgically removed tissue and other breast, kidney, colon, mesothelioma, and lymphoma tissues were provided by the Department of Pathology at CUMC. The accompanying pathology reports indicated the stage of carcinoma in patients according to the AJCC classification. An array (#MNT241, Pantomics, Inc, San Francisco, CA) of multi-normal and tumor tissue sections, consisting of seven common tumor tissues and corresponding normal tissue of brain, breast, colon, liver, lung, prostate, and tonsil on one slide was purchased. A breast disease array (#BRD181, Pantomics, Inc.) consisting of 18 tissues including normal breast tissue, hyperplasia, fibroadenoma, invasive ductal carcinoma, invasive lobular carcinoma, and Paget’s disease on one slide was purchased. A third assay (#UNC241, Pantomics, Inc.) consisting of tonsil, colon, thyroid, thymoma, uterus, placenta, and melanoma tissues in duplicates on one slide was also purchased.
RNA Isolation and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
The flash frozen biopsy tissue was homogenized in Trizol Reagent (Sigma, St. Louis, MO) and the supplier’s instructions were followed for the remainder of the isolation. The quantity of the total RNA was determined using the GeneQuant 1300 spectrophotometer (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). RNA (1μg) was reverse-transcribed into cDNA using the ImProm-II Reverse Transcription System kit (#A3800, Promega, Madison, WI) and the supplier’s instructions were followed with modifications. Nuclease-free water (5.3μL) and MgCl2 (3.2μL) were used for the reverse-transcription reaction mixture which was placed on the heat block at 25°C for 5 minutes, 42°C for 60 minutes, 70°C for 5 minutes, and then 4°C for 5 minutes. Each PCR reaction mixture contained PCR Master Mix (25μL), cDNA (2μL), forward primer (5μL; 1μM), reverse primer (5μL; 1μM), and RNase/DNase Free Water (13μL). The PCR reaction mixtures were placed in the PCR machine heat block and the cDNA was initially denatured at 95°C for 15 minutes. PCR amplification was performed by denaturation for 60 seconds at 42°C, annealing for 60 seconds at 53°C, primer extension for 60 seconds at 72°C. The samples were amplified for an optimized number of cycles and annealing temperatures. For all experiments, 18S was used as an internal control. The PCR product of LRF (NM_015898.2) and 18S were 365bp and 247bp, respectively as deduced from the following sequences:
LRF (forward): 5′ GTCGCAGAAGGTGGAGAAGAAGAT 3′
LRF (reverse): 5′ AGCCGTCTTTCTTGAGGTGTCTCT 3′
18S (forward): 5′ TTGCCATCACTGCCATTAAGGGTG 3′
18S (reverse): 5′ TCTTCACGGAGCTTGTTGTCCAGA 3′
The PCR product was loaded onto an 1.5% agarose gel containing ethidium bromide. After electrophoresis, the gels were photographed under UV light using the UVP Bioimaging System (Upland, CA). Each experiment was performed three times.
The protein concentration of harvested cell lysates from the breast biopsy tissues and cultured breast cells was measured using the Bio-Rad Bradford Protein Assay. The assay was performed in a flat-bottom 96-well microplate according to the supplier’s instructions. The concentrations of the bovine serum albumin solutions that were used as standards for the assay were as follows: 0.05, 0.125, 0.25 and 0.5mg/mL. The samples were mixed thoroughly for three seconds in the microplate mixer and the absorbance was measured at 595nm using a Bio-Rad microplate reader.
Protein solutions were diluted (1:1 v/v) with Laemmli loading buffer with 10% β-mercaptoethanol and heated at 95°C for five minutes before application to the SDS-PAGE gels. A solution containing reference proteins (Precision plus protein dual color) was similarly applied. Electrophoresis was performed at 100V for 1 hour at 25mM Tris buffer containing 192mM glycine and 0.1% (w/v) SDS pH 8.3. The protein was transferred from the gels onto an Immuno-blot PVDF membrane and sandwiched between filter papers. The protein transfer was performed at 400mA for one hour in cold 25mM Tris buffer containing 192mM glycine pH 8.3, methanol (20% v/v), and distilled water (70% v/v). The membranes were incubated overnight in blocking solution [100mL PBS, 5% (w/v) nonfat dried milk, and 0.05% Tween-20] to minimize non-specific protein binding.
The membranes were incubated for one hour at room temperature in a solution of the anti-LRF rabbit polyclonal antibody (1:500) and PBS containing 0.05% Tween-20. The anti-LRF rabbit polyclonal antibody was raised against a peptide derived from the C-terminal region of LRF (NP_056982, amino acids 533–547, Cys-GQEKHFKDEDEDEDV) prepared by Pacific Immunology (Ramona, CA). For all experiments, anti-GAPDH monoclonal mouse antibody (1:2000; NB-1300, Novus Biologicals, Littleton, CO) was used as an internal control. The membranes were incubated in HRP-conjugated secondary antibody solutions (1:1000, anti-rabbit (NB-730H) or anti-mouse (NB-720H), Novus Biologicals) for one hour at room temperature. The antibody-binding was detected using the SuperSignal West Dura Extended Duration Substrates (Pierce, Rockford, IL) and recorded in the UVP Bioimaging System.
Expression of LRF was analyzed using four antibodies specific to four different epitopes of LRF. The LRF antibodies (Pacific Immunology, Novus Biologicals, Abcam, and Santa Cruz Biotechnology) were applied to the tissue sections at dilutions of 1:800 for the brain, tonsil, liver, uterus, placenta, thymus, thymoma, melanoma, and 1:3000 for lung, breast, and colon tissues. Absence of staining (negative control) was demonstrated in control experiments using pre-immunization sera from rabbits immunized with LRF and also when the primary antibody was omitted. hematoxylin and eosin (H&E) staining of sections was used to confirm the clinicopathological status of tissues.
Paraffin sections (4–6μM) were cut from the paraffin-embedded blocks and placed onto Superfrost plus slides (#631-0108, VWR International, Lutterworth, Leicestershire, UK). The sections were deparaffinized and hydrated in xylene and solutions of ethanol gradients [100, 95, 80, and 70 (v/v)]. For immunostaining, antigen was exposed using Target Retrieval Solution (#S1699, DAKO, Carpenteria, CA) by boiling the sections in a steam cooker for 30 minutes. Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide in methanol for 15 minutes. Blocking of non-specific binding sites, incubation with primary antibody (one hour), secondary antibodies, and avidin-biotin complex (ABC) was performed according to the supplier’s instructions from the Vectastain ABC elite kit (PK-6101, Vector Laboratories, Burlingame, CA). Sections were stained with 3,3′-Diaminobenzidine (DAB; SK-4100, Vector Laboratories) and counterstained using hematoxylin for five seconds, dehydrated in solutions of ethanol gradients [70, 80, 95, and 100 (v/v)] and immersed in xylene. Sections were mounted using Permount (#SP-15-100, Fisher Scientific, Pittsburgh, PA) and photographed by an Olympus DP71 camera at 200x or 400x magnification.
Sections were incubated in blocking solution containing phosphate-buffered saline (PBS), 0.25% Triton X-100, 10mg/mL bovine serum albumin (BSA), and 5% normal goat serum (#015-000-120, Jackson Laboratories, West Grove, PA) at room temperature. The cells were incubated in primary antibody solution [anti-LRF (1:1,000) antibody, PBS, 0.1% Triton X-100, 10mg/mL BSA, and 1% normal goat serum] at room temperature. After washing with PBS containing 0.1% BSA three times for five minutes each, a secondary antibody (affinity purified goat anti-rabbit Cy2 antibody, 1:200) was applied to the sections for one hour in the dark to visualize Pokemon-labeled cells (Jackson Immunolabs, West Grove, PA). Negative controls were run in parallel using rabbit pre-immune serum PAC-767 (Pacific Immunology, Ramona, CA) instead of primary antibody or complete omission of primary antibody. Sections were washed with PBS with 0.1% BSA three times for five minutes and dipped into distilled water for two seconds. Fluorescence was preserved by sealing specimens with a solution of equal parts of PBS and glycerol containing 10mg/mL n-propyl gallate, and 1.5mg/mL 4′,6-diamidino-2-phenylindole (DAPI). To prevent the escape of the mounting medium from the coverslips, a single layer of nail polish was placed around the edges. Pictures were taken within 1 hr of the mounting using the Olympus DP71 camera.
Values of all measurements were expressed as the mean ± SE. The Graph Pad Prism 4.0 biochemical statistical package (GraphPad Software, Inc., San Diego, CA, USA) software was used to draw graphs. Values of p<0.05 were considered statistically significant. Nuclear staining for IHC was scored by two independent observers, including a pathologist, in a blinded manner. There was <5% interobserver variation. The semi-quantitative scoring of the nuclear immunostaining was performed as follows: 0 - undetectable, 1+ - weakly positive, 2+ - moderately positive and 3+ - strongly positive.