Primary cell negative controls

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

The wells filled with cells only are referred to as blanks. No primary or secondary antibody will be added to these wells. Instead, these wells will only get PBS. This column will be used to zero the microtiter plate reader. Wells that have PBS instead of the primary or secondary antibody are used as negative controls to assess background. The dilutions in this template are recommendations only. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

Techniques to overcome the lack of a negative (parental) control cell have become available through the use of RNAi, at least in cases where RNAi enters or can be expressed in the primary cells. High-throughput automated imaging systems have helped lessen the need to fully purify cells by allowing analysis at the single cell level. 

Carefully dispense 100 o.L of primary, fluorescein-labeled, antibody, appropriately diluted in BSA-DAB, into the bottom of labeled test tubes. Use 100 pL BSA-DAB in place of diluted antibody for the negative control. Place the tubes on ice until the cells are ready. This can be done while the cells are being centrifuged (see Notes 2 and 3). 

Centrifuge at 400g for 5 min. Discard the supernatant and resuspend the cells in 200 pL of L-15 plus FCS containing the appropriate dilution of the primary antibody. To the negative control sample, add L-15 plus FCS alone. 

Negative-control antibodies (i.e., those that do not react with the experimental cells, IgG from the same species as the primary antibody, or no antibody). 

Develop with Hanker-Yates reagent Development usually is complete by 15 min. However, intensity of development may vary depending on the primary antibody used and the amount of cannabinoid receptor present in tissue. A general approach is to place a negative control (e.g., cells on a cover slip treated with normal rabbit IgG as the primary antibody) side by side with a positive control (e.g., cells transfected with a cannabinoid receptor expression construct). The development reaction is terminated when the positive control qualitatively exhibits a twofold level of staining at the macroscopic level. Alternatively, the staining reaction can be monitored under an inverted cell culture microscope. 

Reuse, S., Maenhaut, C., Dumont, J.E., 1990, Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocj es a positive and negative control by cyclic AMP on c-myc expression, Exp. Cell. Res. 189 33. 

Incubate sections/cells with a blocking solution prepared with NGS and NDS (10 % each) in PBS (use always normal serum from the same species in which secondary antibodies were raised) or 1 % BSA in PBS for 30 min at RT see Note 6). In parallel, run one to two samples as negative controls, where the blocking solution will be applied to sections/cells instead of the primary antibodies, under the same time and temperature conditions see Note 7). 

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