Non-specific Immunostaining by a Rabbit Antibody against Gustducin α Subunit in Mouse Brain

Animals

Nine wild-type male C57BL/6 mice (8 weeks old) from The Jackson Laboratory (Bar Harbor, ME) were used to show gustducin immunoreactivity in the brain and tongue. Six gustducin-knockout mice (Wong et al. 1996) were used as a universal negative control. The procedures and protocols for all animal studies were approved by the Children’s Hospital of Philadelphia and University of Pennsylvania Institutional Animal Care and Use Committees in accordance with international guidelines on the ethical use of animals (National Research Council 1996).

Tissue Processing

The animals were deeply anesthetized with 0.4 ml of 5% chloral hydrate. Intra-cardiac perfusion was performed with saline and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed in the same fixative for 90 min at room temperature. Frontal sections were cut at 50-µm thickness with a Leica VT 1000s vibratome (Leica Microsystems Inc.; Buffalo Grove, IL). To minimize the number of animals sacrificed, the brain sections were collected in series for different immunostaining settings with an interval of 300 µm between two adjacent sections within an identical series.

Tongues from perfused mice were recovered and post-fixed for 3 hr at room temperature. The posterior half, comprising the foliate papillae along both lateral edges and a single circumvallate papilla at the central portion (Mistretta and Baum 1984; Mistretta et al. 1999), was embedded with agarose. Transverse sections at 100 µm in thickness were cut perpendicular to the surface and long axis of the tongue with the Leica vibratome. These tongue slices were collected in three series.

Immunofluorescence Staining

Single and double immunofluorescence staining was performed as previously reported (Xiong et al. 2012). Vibratome sections of the brain and tongue from both wild-type and gustducin-null mice were incubated with an affinity-purified rabbit polyclonal antibody against gustducin α subunit (sc-395; 1:1000 in PBS at a concentration of 0.2 µg/ml; Santa Cruz Biotechnology; Dallas, Texas). This Santa Cruz rabbit antibody recognizes the sequence of 70–120 amino acids of rat gustducin protein from its N-terminus. Visualization was performed with Alexa Fluor ^® 594-conjugated goat anti-rabbit IgG (1:200, Molecular Probes; Grand Island, NY). Primary antibody incubation was applied for 90 min at room temperature and continued overnight at 4°C, and secondary antibodies were subsequently applied for 90 min at room temperature. Immunostained sections were counterstained with Hoechst (Molecular Probes), a nuclear dye to indicate the nuclei of all cells in the tissue for structural verification.

We then tested if a similar staining pattern could be obtained with another affinity purified polyclonal gustducin antibody raised in goat (LS-B4942; 1:500 in PBS at a concentration of 1 µg/ml; Lifespan Biosciences; Seattle WA). This Lifespan Biosciences goat antibody recognizes amino acids 304–318 of human gustducin at its C-terminus. Double immunofluorescence staining was also performed with both rabbit and goat polyclonal antibodies. Consecutively, Alexa Fluor ^® 594-conjugated donkey anti-rabbit IgG (red) and Alexa Fluor ^® 488-conjugated donkey anti-goat IgG (green) were used for the two gustducin antibodies in tongue and brain slices. We also performed double staining in brain sections using the rabbit polyclonal combined with a mouse monoclonal against microtubule-associated protein subtype 2 (MAP2, 1:500; Sigma-Aldrich; St. Louis, MO) or a rat polyclonal against glial fibrillary acidic protein (GFAP; 1:2; Developmental Studies Hybridoma Bank; Iowa City, Iowa). MAP2 is a dendritic marker for neurons from postnatal animals (Bernhardt and Matus 1984) and GFAP, a marker for astrocytes (von Koskull 1984). Combinations of Alexa Fluor ^® 594-conjugated donkey anti-rabbit IgG (red) and Alexa Fluor ^® 488-conjugated donkey anti-mouse IgG (green) or Alexa Fluor ^® 488-conjugated donkey anti-rat IgG (green) were used for visualization.

Control Staining

Peptide blocking and omission of primary antibody were employed as the conventional “negative controls” (Haga and Yoshie 2010). We blocked the rabbit polyclonal with its immunizing peptide (Santa Cruz Biotechnology) before immunostaining. Different ratios of peptide to primary antibody were tested at 5-, 10- or 20-fold by weight (not by moles), reaching a concentration of 1, 2 or 4 µg/ml. The rabbit polyclonal and its blocking peptide were mixed and incubated on shaker for 90 min at RT and then overnight at 4°C. To the first series of brain or tongue sections, the pre-incubated peptide–antibody mixture was applied as a substitute for the original rabbit polyclonal during immunofluorescence staining, as described above. For the second series of sections, the rabbit polyclonal was omitted when performing immunofluorescence staining. A third series of sections were used for regular staining using the original rabbit polyclonal.

Confocal Imaging

Confocal images were acquired with Olympus Fluoview 1000 System (Olympus America, Center Valley, PA), as reported previously (Xiong et al. 2012). Consistent confocal settings were optimized and remained unchanged when imaging slices from different animals. The staining pattern was confirmed in at least three mice. For double immunofluorescence, a sequential imaging module was used to prevent signal leakage from one channel to another. A series of images was stacked to show the general distribution pattern, whereas single confocal images were used to show double staining at higher magnification. For brain structures, the nomenclature of Paxinos and Franklin (2001) was followed.

Fibers Stained using Rabbit Polyclonal Antibody in Wild-type Mouse Brain

We used the Santa Cruz Biotechnology rabbit gustducin antibody to perform fluorescence immunostaining of sections from the whole brain ranging from the olfactory bulb through to the rostral spinal cord. Unlike previous studies showing gustducin-positive neuronal somata or astrocytes in a variety of brain regions (Ren et al. 2009; Dehkordi et al. 2012; Singh et al. 2012), we could not find positive cell bodies in the brain or spinal cord. Instead, we identified numerous gustducin-stained fibers (neuropile) in the nucleus accumbens (Fig. 1A–1C) and periventricular areas (Fig. 1D–1F). Over a period of two years, we tested three lots of the rabbit gustducin antibody and acquired the same staining pattern. In the nucleus accumbens, gustducin staining was seen solely in its core region (Fig. 1A). In the periventricular areas, gustducin staining was predominantly seen surrounding the ventral third ventricle (Fig. 1D). A small number of gustducin-stained fibers were distributed along the ventromedial bank of the lateral ventricle and the dorsal third ventricle. Fiber staining was occasionally distributed along the aqueduct but not seen around the fourth ventricle and the central canal of the spinal cord. Under high magnification, these stained fibers displayed a bead-like morphology (Fig. 1C and 1F; Insets). Double fluorescence staining demonstrated that these gustducin-stained fibers were not co-stained by either MAP2 (Fig. 2A–2C) or GFAP (Fig. 2D–2F), confirming that they do not belong to neuronal dendritic arbors or astrocytic fibers.

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Figure 1.

Gustducin-stained fibers in nucleus accumbens and periventricular areas in wild-type mouse. Rabbit polyclonal antibody against gustducin α subunit (GNAT3 protein) was used. (A) Gustducin staining in the nucleus accumbens and ventral part of the lateral septal nucleus (LSV). ac, anterior commissure; CPu, caudatus-putamen; LV, lateral ventricle. (B) Higher magnification of gustducin staining in the nucleus accumbens in (A). (C) Higher magnification of stained components in (B) exhibiting a bead-like morphology (inset). (D) Gustducin staining in the periventricular area surrounding the ventral third ventricle (3V). SCh, suprachiasmatic nucleus. (E) Higher magnification of gustducin stained fibers in (D). (F) Bead-like appearance (inset) of gustducin-stained fibers in (E) under higher magnification. Arrows in (C) and (F) indicate a single fiber highlighted in the Inset. Scale (A and D), 50 µm; (B and E), 20 µm; (C and F), 10 µm; insets, 10 µm.

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Figure 2.

Gustducin-stained fibers may be axonal structures. (A–C) Co-staining of rabbit anti-gustducin (A, red) and mouse anti-microtubule associated protein subtype 2 (MAP2; B, green) showed that these gustducin-stained fibers were not MAP2-positive (C). (Inset) Higher magnification of a single fiber in C (arrow) stained with gustducin only. Some gustducin-stained neuropiles were present in a very close contact with MAP2-positive dendrites (yellow). (D–F) Co-staining of rabbit anti-gustducin (D, red) and rat anti-glial fibrillary acidic protein (GFAP; E, green) showed that these fibers were not GFAP-positive (F). (Inset) Higher magnification of the single fiber in F (arrow) stained by gustducin only. Scale (A–F), 10 µm; insets, 5 µm.

Control Staining in Tongue Slices

To verify the specificity of the rabbit gustducin antibody, we performed double immunofluorescence staining in tongue slices (Fig. 3) using both rabbit and goat gustducin antibodies. In wild-type mice, taste cells were simultaneously stained with both rabbit (red) and goat (green) antibodies, showing a perfect co-staining (Fig. 3A, yellow). In gustducin-knockout mice, tongue slices were not stained by either of the two antibodies (Fig. 3D).

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Figure 3.

Gustducin and control staining in the circumvallate papilla of the tongue from wild-type (A–C) and gustducin knock-out mice (D). (A) Double staining with both gustducin antibodies raised from rabbit (red) and goat (green) showed perfect co-staining (yellow). (B) Pre-incubation with immune peptide for the rabbit antibody (Pep) completely blocked immunostaining from this antibody, leaving goat antibody staining (green) unaltered. (C) Omission of both antibodies (-Abs) resulted in no staining. (D) Double staining with both gustducin antibodies showed no positive staining in the tongue from gustducin knockout mice. Scale, 20 µm.

We then performed peptide blocking in tongue slices from wild-type mice using different ratios of the immune peptide to the rabbit gustducin antibody (5:1, 10:1 and 20:1). Complete blocking of the rabbit gustducin antibody staining could be reached only at the ratio of 20:1 (peptide concentration at 4 µg/ml) in tongue slices, leaving taste cells stained solely by the goat antibody (Fig. 3B, green). On the other hand, omission of both rabbit and goat polyclonal antibodies resulted in no staining in tongue slices (Fig. 3C), suggesting that the secondary antibodies gave no detectable non-specific staining in the present protocol.

Control Staining in Brain Slices

Because gustducin immunostaining was completely abolished in the tongue slices (Fig. 3D) from gustducin knockout mice (Wong et al. 1996), we expected negative staining in the brain slices from these mice as well. To our surprise, we consistently detected a high density of stained fibers along the ventral third ventricle and nucleus accumbens core. A small number of stained fibers were also seen in periventricular areas along the lateral ventricle, dorsal third ventricle and aqueduct. Similarly as in wild-type mice, no gustducin-stained neuronal somata or astrocytes were identified in brain slices from gustducin knockout mice. We then performed double immunostaining with both rabbit and goat antibodies to mouse brain slices. The goat gustducin antibody (green) did not stain the periventricular and accumbens fibers in wild-type (Fig. 4A and 4B) or knockout mice (Fig. 4D and 4E), leaving a single staining of the rabbit gustducin antibody (Fig. 4, red). A close comparison showed that there was no obvious difference in the general distribution pattern of these fibers in the gustducin-knockout mice (Fig. 4D and 4E) as compared with the wild-type mice (Fig. 4A and 4B). Under high magnification (Fig. 4; insets), stained fibers in knockout mice (Fig. 4D and 4E) exhibited a similar morphology to that of wild-type mice (Fig. 4A and 4B). Finally, conventional control experiments were performed in brain slices. We found that fiber staining by the rabbit gustducin antibody could not be completely blocked (Fig. 4c, red) even at the concentration 4 µg/ml of blocking peptide. As anticipated, omission of both rabbit and goat gustducin antibodies resulted in no staining in brain slices (Fig. 4F).

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Figure 4.

Fibers stained by the rabbit antibody (red) are not stained by the goat antibody. Rabbit antibody-stained fibers in the nucleus accumbens (A) and periventricular area (B) from wild-type mouse. (C) Pre-incubation with immune peptide for the rabbit antibody could not block immunostaining from this antibody in the brain. Rabbit antibody-stained fibers in nucleus accumbens (D) and periventricular area (E) from gustducin-knockout mouse. A similar distribution pattern and bead-like morphology (insets) are seen from wild-type (A and B) and knockout (D and E) mice. Arrows indicate the position of the higher magnification images (insets). (F) Omission of both primary antibodies resulted in no staining at all. Scale (A–F), 20 µm; insets, 20 µm.