Isotype matched

Isotype-matched unconjugated or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers. 

For the negative control, primary antibody is replaced with an irrelevant, isotype-matched antibody. This step controls nonspecific binding of the secondary antibody. The... 

UPCIO (Antimyeloma antibody used as an isotype-matching nonspecific control) (ICN Pharmaceuticals). 

Add diluted mouse anti-CCL21 and rabbit anti-CCR7 antibodies (in the Antibody Diluent provided with the PLA Probe kit) to the tissue. As negative controls, isotype-matched control IgG antibodies were added at same dilution. 

A representative image of PLA signal from tumor tissues labeled with either isotype-matched control IgGs or specific CCR7 and CCL21 antibodies are shown in Fig. 5. Both images were from the same region on two sequential slides of the same LLC tumor tissue. 

Coronal cryostat sections (10 pm thick) are cut after cryoprotec-tion and processed for indirect IMF. Sections are first incubated for 60 min with 10 % NGS or 10 % normal donkey serum in PBS to block nonspecific binding. They are then incubated with antibodies diluted according to manufacturers recommendations 1 h with primary antibody and then 1 h with secondary antibody diluted 1 100. Double IMF is performed according to standard protocols [48]. Mounting media contain DAPI to stain nuclei. Negative controls are performed each time immunostaining was done and consisted of preincubation with PBS, substitution of nonimmune isotype-matched control antibodies for the primary antibody, and/or omission of the primary antibody. Further details can be found in [48]. 

Positive selection is not recommended as these procedures may induce signaling pathways in CLL cells due to antibody/antigen interactions. Purity of CLL cells is assessed by flow cytometry using anti-CD19, anti-CD5, and matching isotype control... 

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