Digoxigenylated primary antibodies for sensitive dual-peroxidase labelling of neural markers

Abstract

This study extends the application of the digoxigenin-anti-digoxigenin (DIG) technique to immunocytochemistry by using digoxigenin-tagged primary antibodies. Certain features of this technique when applied to non-radioactive in situ hybridization, such as the absence of endogeneous digoxigenin immunoreactivity in animal tissues, seem to be advantageous also for its application to immunocytochemistry. Thus, the present work is focused on dual-peroxidase staining experiments based on digoxigenylated antibodies directed against glial fibrillary acidic protein, parvalbumin, and calbindin, in a straightforward combination with conventional cytochemical methods. The protocols include the concomitant detection of two antigens, for which only primary antibodies from one animal species are available, with differently haptenized antibodies (e.g., biotinylated anti-calbindin and digoxigenylated anti-parvalbumin). The versatility of the DIG technique is exemplified by the combination of lectin and immunocytochemical procedures for the detection of astrocytes and microglia, and the simultaneous visualization of perineuronal nets and parvalbumin-containing neurons in the rat brain.

Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling

“…These include: (1) immunofluorescence or immunoenzymatic doublestaining procedures with two primary antibodies from the same species, (2) detection of immunoprecipitated antigen on western blots when simultaneous visualization of the precipitating IgG has to be avoided, and (3) quantification of immunochemically defined proteins in crude mixtures using sandwich-type ELISA assays. Antibody haptenylation for immunocytochemical double staining has been described by several authors (van der Loos et al 1987;Behringer et al 1991;Härtig et al 1994Härtig et al , 1995, and suppliers usually provide protocols for their activated haptens. However, combined affinity purification and haptenylation has not been desribed so far.…”

Cracking Down on Inhibition: Selective Removal of GABAergic Interneurons from Hippocampal Networks

“…For immunoperoxidase staining, sections were applied to a streptavidin-biotin method and Ni-enhanced 3,3Ј-diaminobenzidine (DAB-Ni) as described previously (Härtig et al, 1995). The following primary antibodies were used: mouse antiparvalbumin (1:4000; Swant), guinea pig anti-parvalbumin (1:2000; Synaptic Systems), mouse anti-glutamic acid decarboxylase 67 kDa isoform (GAD67; 1:5000, Millipore Bioscience Research Reagents) and guinea pig anti-VGAT, N terminus (1:500, Synaptic Systems) (Martens et al, 2008).…”

Rabbit forebrain cholinergic system: Morphological characterization of nuclei and distribution of cholinergic terminals in the cerebral cortex and hippocampus

“…After extensive rinsing, sections were reacted with biotinylated goat anti-mouse IgG (Jackson Immunoresearch; 2.5 g/ml in TBS-BSA) for 1 hour. Thereafter, tissue samples were processed according to a visualization protocol identical to that described for the immunodetection of ChAT (Hä rtig et al, 1995).…”

“…Subsequently, sections were thoroughly rinsed and exposed to biotinylated donkey anti-sheep IgG [highly cross-reacting with goat IgG (Jackson Immunoresearch); 1:500 in TBS containing 2% bovine serum albumin (TBS-BSA)] for 1 hour. Finally, sections were processed with preformed complexes of streptavidin and biotinylated peroxidase and stained with nickel-enhanced 3,3Ј-diaminobenzidine as chromogen (Hä rtig et al, 1995).…”