Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Abstract

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.

Intracellular Aβ pathology and early cognitive impairments in a transgenic rat overexpressing human amyloid precursor protein: a multidimensional study

“…Sections were incubated in 0.3% hydrogen peroxide in PBS for 20 min, washed in PBS-T (0.01 M phosphate-buffered saline, 0.2% Triton X-100) and blocked 1 h with 10% normal goat serum (NGS) in PBS-T. To examine the evolution of the AD-like amyloid pathology we incubated sections with the monoclonal antibody McSA1 [36] (MediMabs, Montreal, Canada) at 1:4000 in PBS-T with 5% NGS overnight at 4°C. The following day, the sections were washed in PBS-T and incubated with a goat anti-mouse secondary antibody (MP Biochemicals, Canada) 1:100 in PBS with 5% NGS for 1 h. The sections were washed in PBS and incubated for 1 h with a mouse anti-peroxidase monoclonal antibody [37] (1:30) pre-incubated with horseradish peroxidase (5 μg/ml) in PBS (MAP kit, Medimabs, Canada). Stainings were developed with 0.06% 3,3′-diaminobenzidine (Sigma-Aldrich, USA) and 0.01% hydrogen peroxide (Sigma-Aldrich, USA) in PBS and then mounted on subbed slides.…”

Reorganization of Cholinergic Terminals in the Cerebral Cortex and Hippocampus in Transgenic Mice Carrying Mutated Presenilin-1 and Amyloid Precursor Protein Transgenes

“…Before immunostaining, brain sections were incubated in 0.5% H 2 O 2 for 30 min, followed by a treatment of 3% bovine serum albumin (Sigma) for 60 min at room temperature. Sections were then incubated with the mouse anti-A␤ antibody (1:1500) overnight at 4°C, followed by a 1 hr incubation with goat anti-mouse IgG (1:50; IC N Biochemicals, Montréal, Québec, C anada) and a 1 hr incubation with monoclonal mouse anti-peroxidase antibody (1:30; Medicorp; Semenenko et al, 1985) in the presence of 5 g /ml horseradish peroxidase (Sigma, type IV) at room temperature. After 15 min of DAB treatment, H 2 O 2 was added, and the reaction was continued for 4 min.…”

Parasympathetic nerve fibers invade the upper dermis following sensory denervation of the rat lower lip skin

“…The anti-D␤H antibody was generated against rat D␤H and has been characterized as highly specific (Mazzoni et al, 1991). The sections were then incubated for 2 hours in goat antimouse IgG (1:50; American Qualex, San Clemente, CA) preabsorbed in fixed rat lip tissue for 8 hours at 4°C (Ruocco et al, 2000) and then treated for 1 hour with a mouse monoclonal anti-HRP antibody (Seralab, Leicestershire, UK; Semenenko et al, 1985) to which 5 g/ml HRP had been previously added. After further washing, D␤H immunoreactivity was revealed by reacting the sections with DAB and H 2 O 2 .…”