Methodological Considerations

With standard DAB / peroxidase immunocytochemistry, a conclusive analysis of the neuropil labeling observed with anti-Agm antibodies has proven difficult, at least in our hands. The combination of pre-embedding hapten-amplification using CARD and post-embedding visualization of the deposited haptens has been previously suggested as a sensitive approach suited for the detection of rare antigens (Humbel et al., 1998). However, only labeling of cultured cells has been reported to date, which is clearly different from labeling of brain tissue in terms of antibody penetration and background labeling. This approach, here referred to as virtual pre-embedding (VIRP), indeed proved to offer a useful alternative for ultrastructural pre-embedding labeling in rat brain tissue sections. With this technique, Agm-like immunoreactivity in dendritic spines was not only observed more frequently and reliably, but also was detected in some presynaptic terminals at spine synapses. Thus, the CARD-based VIRP procedure offers an increased sensitivity paired with the opportunity to locate regions of interest in semithin sections parallel to thin sections used for ultrastructural analysis. Moreover, the particulate nature of the final VIRP reaction product also offers an increased spatial resolution when compared with DAB-labeling.

Virtual Pre-embedding procedure

Pre-embedding Deposition of Haptens

Freshly perfused brain tissue was sectioned at a thickness of 50 μm using a vibratome. Sectionswere rinsed in PBS, followed by incubation in 20% sucrose in 0.1 M phosphatebuffer twice for 10 min. Subsequently, they were transferred on a plastic support and freeze-thawed using liquid nitrogen(Lujan et al., 1997). After washing in PBS, sections were treated with 0.1% sodium borohydride in PBS for 15 minutes. After washing in PBS, sections were pre-incubated for 30 minutes in 10% normal goat serum in PBS, containing 0.05 % triton for permeabilization and 0.05% phenylhydrazine. Subsequently, the sections were incubated for at least 36 hours at 4 °C in the primary antibody solution (rabbit-anti-agmatinase 1:5.000), containing 10% NGS in PBS, 0.05% triton, 0.1% sodium azide and 0.01% thiomersal. Sections were rinsed twice for 20 and 40 minutes in PBS, followed by pre-incubation with PBS-Albumin (PBS-A, 2% w/v bovine serum albumine in PBS) for 1 hour and incubation over night at 4 °C with the biotinylated secondary antibody (biotin-goat-anti-rabbit, diluted 1:2.000 in PBS-A). After two washing steps in PBSfor 20 and 40 minutes and pre-incubation in PBS-A for 1 hour, sections were incubated overnight at 4 °C with ELITE ABC (Vector, 1:200) in PBS-A. After rinsing in PBS three times for 10, 20 and 30 minutes, sections were pre-incubated for 15 minutes with 10 µM of Tetramethylrhodamine-tyramide (TMR-T) diluted in CARD-solution (50 mM Tris-Buffer, 10 mM imidazole). TRM-T was deposited by adding hydrogen peroxide (0.0015% final concentration) for 15 minutes.

Epoxy embedding

Sections were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 10 minutes and washed in phosphate buffer. Subsequently, they were dehydrated in a graded series ofethanol including a block stainingwith 2% uranyl acetate in 70%

ethanol for 30 minutes. Sections were transferred to 100% propylene oxide twice for 10 minutes, followed by incubation in a 1:1 mixture of propylene oxide and araldite for 10 min and pure araldite for 20 min. Sections were then transferred to freshly preparedresin and flat-embedded.The specimen were then polymerized for 24h at 60°C.

Preperation of semithin and ultrathin sections

Semithin (500 nm) and ultrathin sections (60-70 nm) were cut using a diamond

knife (Diatome, Switzerland) and an ultramicrotome (Reichert Ultracut S, Leica, Germany).Semithin sections were dried at 70 °C to aminosilane-coated slides. Ultrathin sections werecollected on 0.75% pioloform-coated 200 to 300 mesh nickel grids.

Post-embedding immunocytochemistry on semithin sections

Sections were incubated in methanolate etching solution (1 M Sodium methoxide in a 2:1 mixture of methanol and toluene)for 10 minutes, followed by rinsing in a1:1 mixture of methanol and toluene for 5 minutes and twice in acetone for 5 minutes. Afterrinsing with distilled water, slides were transferred for a few minutes in 100 mM acetatebuffer (pH 5). Afterwards, sections were incubated in 2% hydrogen peroxide for 5 minutes,transferred again into acetate buffer for several minutes and finally rinsed in PBS.Slides were then transferred into a humid chamber. All following steps were performed atroom temperature. Sections were pre incubated in 10% NGS for 30 minutes, followed byincubation with rabbit-anti-tetramethylrhodamine (RaTMR) diluted 1:7.500in 10% NGS over night. Afterrinsing twice for 5 and 10 minutes in PBS, sections were pre-incubated in PBS-A for 15minutes and then with B-GaR 1:1.000 for 4 hours.Having rinsed again twice for 5 and 10 minutes in PBS and pre-incubation in PBS-A, sectionswere incubated with ELITE ABC diluted 1:200 in PBS-A for 1 hour. After washing twice in PBS for 5 and 10 minutes, sections were pre-incubated in DAB-solution for 15 minutes. Visualization with DAB was achieved by adding ANS (0,3 % finalconcentration) and hydrogen peroxide (0,0015% final concentration) for 15 minutes.

Post-embedding immunocytochemistry on ultrathin sections

All steps were performed at room temperature. Grids were placed on 50 μl-droplets on a sheet of parafilm. First, grids were incubated for 7 min each in 1% periodic acid and 1% sodium metaperiodate. Subsequently, the grids were jet-rinsed with distilled water. Grids were then incubated in TBSX (50 mM TRIS, 0.01% Triton X-100, 139 mM Sodium hydrochloride)for 10 minutes, followed by pre-incubation in Block 1 (5% normal goat serum, 2% bovine serum albumine, in TBSX) for 30 minutes. RaTMR was diluted 1:500 in Block 1 and the grids were incubated over night. The next day, sections were jet rinsed with TBSX twice, pre-incubated in Block 2 (2% Bovine serum albumine, in TBSX)for 10 minutes and incubated in the secondary antibody solution (10nm gold goat-anti-rabbit, diluted 1:50 in Block 2) for 90 minutes. Grids were then jet-rinsed twice in TBSX, once in distilled water, and air-dried.

Staining of ultrathin sections

Grids were double stained with 2% uranyl acetate in 70% ethanol for 2 minutes and

lead citrate for 20 seconds.

Humbel BM, de Jong MD, Muller WH, Verkleij AJ. 1998. Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers. Microsc Res Tech 42(1):43-58.

Lujan R, Roberts JD, Shigemoto R, Ohishi H, Somogyi P. 1997. Differential plasma membrane distribution of metabotropic glutamate receptors mGluR1 alpha, mGluR2 and mGluR5, relative to neurotransmitter release sites. J Chem Neuroanat 13(4):219-241.