Immunohistochemistry (IHC) for Light and Transmission Electron Microscopy (LM and TEM)

Male rats of 270 ± 30 g were deeply anesthetized with an overdose of sodium pentobarbital (63 mg/kg, Sedalpharma, Mexico) and perfused transaortically with 0.9% saline followed by cold fixative containing 4% of paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4) plus 15% v/v saturated picric acid for 15 min (0.05% of glutaraldehyde was added to fixative for the samples intended for TEM). Brains were immediately removed, blocked, then thoroughly rinsed with 0.1 M PB until they were clear of fixative. The brains were then sectioned using a Leica VT 1000S vibratome, at 70 μm thickness in the horizontal plane.

For LM IHC, non-specific binding of the secondary antibodies was minimized by incubating sections containing LC with 20% normal donkey serum (NDS) in Tris-buffered (0.05 M, pH 7.4) saline (0.9%) plus 0.3% of Triton X-100 (TBST) for 1 h at room temperature (RT). The sections were then incubated with the following primary antibodies: rabbit anti-AVP antibody (kind gift of Dr. Ruud M. Buijs; Buijs et al., 1989), mouse anti-AVP antibody (kind gift of Dr. Hal Gainer; Alstein et al., 1988), rabbit anti-AVP (Abcam, ab39363, for Figure 1B) sheep anti-tyrosine hydroxylase (TH) [Abcam, ab113; guinea pig anti-VGLUT2 (Frontier Institute, VGLUT2, GP-Af810)]. The next day, the sections were washed with TBST for 30 min after which they were incubated at RT in a cocktail of an appropriate mixture of secondary antibodies, conjugated with Alexa Fluor 488, Alexa 594 and indocarbocyanine (Cy5), all provided by Jackson ImmunoResearch, for 2 h. The sections were washed in TBST and were mounted on glass slides, air dried and coverslipped using Vectashield mounting medium (H-1000, Vector Laboratories Inc.).

AVP–VGLUT2 immunopositive (AVP+/VGLUT2+) fibers establish Gray type I (asymmetric) synapses onto tyrosine hydroxylase immunopositive (TH+) dendrites within the LC. (A,B) Confocal photomicrographs showing AVP+ fibers making contacts with TH+ dendrites (A) and the co-localization of AVP+/VGLUT2+ segments (B, two Herring body-like bodies were indicated by arrow heads). (A) AVP reaction was made using the antibody gifted from H. Gainer laboratory and image was taken at 0.5 AU (Airy Units, using Leica SP5 confocal system) optical section thickness of around 0.596 μm. This measure helps to improve the optical resolution to visualize the contact points in a strongly labeled tissue. The panel B was from mouse LC tissue using a different antibody (Abcam ab39363) and taken at 1 AU (optical section thickness was around 0.892 μm). (C) Photomicrograph taken from a pre-embedding immunoreaction of trimmed resin capsule surface (the “pyramid,” osmicated losing the purple color under LM), containing LC prepared for electron microcopy (EM), using DAB/VIP (Very Intense Purple) double peroxidase-chromogen immunostaining for electron microscopy. AVP+ fibers were evidently making contact with TH+ dendritic segments indicated by arrowheads. The inserts are TEM micrographs from serial samples of the region indicated by rectangle area in C. The image shows an AVP+ axon with a terminal (depicted in four serial sections) containing AVP+ dense-core vesicles (dcv, indicated with green arrowhead), establishing a Gray-type I synapse onto a TH+ dendrite (TH is demonstrated by granular labeling produced by VIP reagent at electron microscopy level, yellow arrowheads). Postsynaptic density (PSD), a TEM feature of a Gray type I synapse, which is generally indicative of a glutamatergic synapse, are indicated by green arrows. (D) TEM micrograph showing one segment of the same AVP+ profile (DAB-nickel labeling) coursing in parallel with two TH+ (VIP labeling) dendrites. Note that the VIP labeling for dendrite (white arrowhead) was weaker than for axonal segment (black arrowhead). (E) An Herring body-like body (large axonal varicosity), an anatomical feature of the vasopressinergic magnocellular neurosecretory neurons (white asterisks of panel B,E) examined under EM. Scale bars: 400 nm unless stated otherwise.

For double peroxidase-chromogen immunostaining and electron microscopy (TEM), brain sections containing LC, were cryoprotected with 10% and then 20% sucrose in PB (under gentle agitation until the sections sank). Permeability of the tissue was then enhanced by rapidly freezing and thawing sections using liquid nitrogen. Sections were then thoroughly rinsed with PB 0.1 M and non-specific secondary antibody binding was minimized with 20% NDS in TBS for 1 h at RT. The sections were then incubated with rabbit anti-AVP and sheep anti-TH antibodies (see above) in TBS plus 1% NDS for 48 h at 4°C with gentle agitation. After rinsing in TBS, incubation was continued with first secondary antibody swine anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (1:100, Dako P021702, Copenhagen, Denmark), in TBS containing 1% of NDS, overnight at 4°C. Sections were then rinsed and peroxidase enzyme reaction was carried out using the chromogen 3,3′-diaminobenzidine (DAB, 0.05%, Electron Microscopy Sciences) and hydrogen peroxide (H₂O₂, 0.01%) as the substrate. The reaction end product in some sections was intensified with nickel. Subsequently, sections were incubated with 2nd secondary antibody, biotinylated goat anti sheep antibody (Jackson ImmunoResearch Laboratories) and then incubated with Vectastain standard ABC kit [VECTASTAIN^®Elite^®ABC HRP Kit (Peroxidase, Standard), Cat. No: PK-6100, Vector Laboratories, Burlingame, CA, United States]. The TH immunoreactivity was then visualized by using a Vector-VIP (very intense purple) peroxidase substrate kit [VECTOR^®VIP Peroxidase (HRP) Substrate Kit; Vector Laboratories]. This procedure yields a reaction product that appears purple in the light microscope and granular or particulate in the electron microscope. Sections were then post-fixed with 1% osmium tetroxide in 0.1 M PB for 1 h and dehydrated through a series of graded alcohols (including 45 min of incubation in 1% uranyl acetate in 70% ethanol), then transferred to propylene oxide, followed by Durcupan ACM epoxy resin (Cat. No. 100503-434, Electron Microscopy Sciences). Sections were flat embedded on glass microscope slides, and the resin was polymerized at 60°C for 2 days. After removing the coverslip, LC containing regions, identified by TH immunoreactivity, were sectioned and carefully re-embedded in capsules in Durcupan resin. Ultrathin serial sections (70 nm) were prepared with an ultramicrotome, collected on pioloform-coated single slot grids and examined with a Philips CM100 transmission electron microscope. Digital electron micrographs were obtained with a digital micrograph 3.4 camera (Gatan Inc., Pleasanton, CA, United States).