Comparison of Peroxidase‐Anti‐Peroxidase and Avidin‐Biotin‐Peroxidase Complex (ABC) Immunohistochemical Staining Procedures for Tryptophan Hydroxylase in Rat Brain

Abstract

The neurotransmitter serotonin (5‐hydroxytryptamine; 5‐HT) plays a key role in sleep, feeding behavior, motor activity and, importantly, mood and the mechanism of action of antidepressant drugs. Immunohistochemistry for tryptophan hydroxylase (TpOH), the synthetic enzyme for 5‐HT, can be used to identify 5‐HT neurons in the central nervous system. As part of a project examining 5‐HT in brain function in the rat, we compared 2 indirect methods of visualizing primary antibodies against TpOH, a peroxidase‐anti‐peroxidase (PAP) method and the more sensitive avidin‐biotin‐peroxidase (ABC) procedure.A cryostat microtome was used to cut 40 micron sections from brains from rats transcardially perfused with 0.9% saline and 4% p‐formaldehyde in 0.1 M phosphate buffer (PB). Brains were post‐fixed 24 h in p‐formaldehyde and cryoprotected in 20% sucrose in 0.1 M PB. Sections were washed in phosphate‐buffered saline (PBS) before and between all steps. Tissues were treated with 0.4% H2O2 in methanol for 20 min to quench endogenous peroxidase activity and 1% sodium borohydride in 0.1 M PB for 30 min to enhance permeability of the tissue and improve staining quality. Sections were then treated with 1% normal donkey (PAP method) or horse (ABC method) serum in PBS with 0.2% Triton X‐100 detergent for 1 h and then 0.8% bovine serum albumin in PBS for 40 min. Tissues were incubated for 48 h in mouse monoclonal anti‐TpOH antibody (Sigma, 1:1000) in PBS with 0.3% Triton X‐100 and serum. Some sections were incubated without the primary antibody (omit negative controls). For the PAP protocol, tissues were treated with donkey anti‐mouse antibody (Jackson; 1:100) and then mouse PAP (Jackson; 1:400), both in PBS with 0.3% Triton X‐100 for 1 h. For the ABC protocol, tissues were treated with biotinylated horse anti‐mouse antibody and then avidin‐biotin‐peroxidase complex (Vector Elite kit), both in PBS with 0.3% Triton X‐100 and serum for 1 h. All sections were reacted with 0.0005% diaminobenzidine with 0.0001% H2O2 for 45 – 120 sec and mounted on gelatin‐coated slides that were coverslipped with DPX. Stained and negative control sections from both methods were examined using standard light microscopy.Staining using the PAP method was faint, homogenous and only slightly darker than negative controls. Individual neurons could not be identified. Increasing concentrations (1:500, 1:250, 1:100) of anti‐TpOH antibody had no effect on specific labeling but slightly increased background staining. This was surprising because the exact same reagents and PAP procedure has produced outstanding staining of tyrosine hydroxylase in brainstem catecholamine neurons (e.g., Muma et al., Exp. Neurology, 168:135–143, 2001). In contrast, labeling of TpOH using the ABC method was excellent. TpOH immunoreactivity was highly visible in the somata and primary dendrites of neurons in the dorsal and median raphe nuclei. These results add to and amplify the enhanced staining quality of the ABC procedure over the PAP method in formaldehyde‐fixed rat brain sections.Support or Funding InformationSupported by the School of Biological Sciences and the Department of Psychology, Illinois State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.