γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

Abstract

Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the γ-aminobutyric acid type A (GABA_A) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3β-hydroxysteroid dehydrogenase (3β-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABA_A receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3β-HSD-positive neurons also contain GABA_A receptor α₃ and β₂/β₃ subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA_A receptor agonist muscimol but was not affected by the GABA_B receptor agonist baclofen. The selective GABA_A receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABA_A receptor α₃ and β₂/β₃ subunits. Our data also demonstrate that GABA, acting on GABA_A receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3β-HSD and cytochrome P450_C17 (17α-hydroxylase).

There is now clear evidence that the brain has the capability to synthesize bioactive steroids called “neurosteroids” (1). Immunohistochemical and in situ hybridization studies have revealed the presence of several steroidogenic enzymes in glial cells and/or neurons (2). Concurrently, biochemical investigations have shown that brain explants or cultured neural cells can synthesize in vitro various regulatory steroids (3, 4). In particular, it has been found that frog hypothalamic neurons express 3β-hydroxysteroid dehydrogenase (3β-HSD), a key enzyme of the steroid biosynthetic pathway, and it has been demonstrated that frog hypothalamic tissue can convert the steroid precursor pregnenolone into various bioactive metabolites, including 17-hydroxypregnenolone (17OH-Δ⁵P), progesterone (P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA), and dihydrotestosterone (5α-DHT; refs. 5, 6).

The effects of neurosteroids on nerve cells are modulated through several distinct categories of receptors. Neurosteroids, like other steroid hormones, can act at the transcriptional level via nuclear receptors (7). Neurosteroids may also interact with plasma membrane G-protein-coupled receptors (8, 9). However, most of the actions of neurosteroids appear to be mediated through γ-aminobutyric acid type A (GABA_A) receptors (10, 11). For instance, the effects of GABA on the GABA_A receptor are allosterically modulated by progesterone and deoxycorticosterone metabolites such as allopregnanolone, pregnanolone, and tetrahydrodeoxycorticosterone (1, 11).

Although neurosteroids are potent regulators of neuronal activities (11, 12), little is known concerning the control of steroid biosynthesis in the brain. In particular, the possible involvement of GABA in the regulation of steroid-producing neurons has received little attention (13, 14). In the present report, we have searched for the presence of GABA_A receptors in 3β-HSD-containing neurons in the frog hypothalamus, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants.

Materials and Methods

Animals.

Adult male frogs (Rana ridibunda) of about 30–40 g body weight were purchased from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). The animals were maintained under artificial illumination (light on from 06:00 to 18:00) in a temperature-controlled room (8 ± 0.5°C), for at least 1 week before use. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators. To limit possible variations of neurosteroid biosynthesis caused by circadian rhythms (15), all animals were killed between 09:30 and 10:30.

Antibodies.

The antiserum against human placental type-I 3β-HSD was raised in rabbit (16). Polyclonal antibodies directed against the α₃ subunit of the rat GABA_A receptor were raised in rabbit (17, 18). The mouse monoclonal antibodies against the β₂/β₃ subunits of the mammalian GABA_A receptor (clone bd17) were purchased from Roche Molecular Biochemicals (Meylan, France). Alexa-488-conjugated goat anti-mouse γ-globulins (GAM_S/Alexa-488) were from Molecular Probes. FITC-conjugated goat anti-rabbit γ-globulins were from Nordic (Tilburg, the Netherlands). Texas Red-conjugated donkey anti-rabbit γ-globulins (DAR_S/TXR) were from Amersham International.

Immunofluorescence Procedure.

Animals were anesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester (MS222) and perfused transcardially with 50 ml of PBS (pH 7.4). The perfusion was carried on with 50 ml of either Bouin's fixative or McLean's fixative. The brains were postfixed overnight at 4°C, embedded in Tissue Teck (Reichert-Jung), frozen at −80°C, and cut in the frontal or sagittal plane in a cryostat (6-μm-thick sections). Consecutive tissue sections were incubated overnight at 4°C in a humid atmosphere with the 3β-HSD antiserum (1:100), the polyclonal antibodies against the α₃ subunit of the GABA_A receptor (5 μg immunoglobulins/ml), or the monoclonal antibodies against the β₂/β₃ subunits of the GABA_A receptor (10 μg immunoglobulins/ml) in PBS containing 0.3% Triton X-100 and 1% BSA. The sections were rinsed in three successive baths of PBS and then incubated for 90 min at room temperature with DAR_S/TXR (1:50), GAM_S/Alexa-488 (20 μg/ml), or FITC-conjugated goat anti-rabbit γ-globulins (1:100). Other brain sections were incubated with a mixture containing the 3β-HSD antiserum and the anti-GABA_A receptor β₂/β₃ subunit immunoglobulins, and the immunoreactivity was revealed with DAR_S/TXR and GAM_S/Alexa-488. Finally, the sections were rinsed in PBS and mounted with PBS-glycerol (1:1).

The preparations were examined by using a confocal laser scanning microscope equipped with a diaplan optical system and an argon/krypton ion laser (excitation wavelengths: 488/568/647 nm; Leica, Heidelberg, Germany). Dual-channel confocal laser scanning microscopic analysis was performed using a band-pass filter (λ = 535 ± 7 nm) for detection of FITC or Alexa-488, and a long-pass filter (λ = 610 nm) for detection of TXR.

The specificity of the immunoreaction was controlled either by substituting the primary antisera with PBS or by preincubating the antibodies against 3β-HSD with purified human type I 3β-HSD (10⁻⁶ M) and the antibodies against the GABA_A receptor α₃ subunit with the synthetic peptide hapten (16 μg/ml).

Pulse-Chase Technique.

Conversion of [³H]Δ⁵P into progesterone, 17OH-Δ⁵P/5α-DHT, 17OH-P, and DHEA was studied using a pulse-chase technique as previously described (5). For each experiment, the hypothalami of four frogs were rapidly dissected, and each hypothalamus was cut into two pieces. The tissue fragments were preincubated for 15 min in 1 ml of Ringer's solution (pH 7.4). The hypothalamic slices were incubated at 24°C for 2 h in 500 μl of Ringer's medium containing 4% propylene glycol and 10⁻⁶ M [³H]Δ⁵P, in the absence or presence of test substances. At the end of the incubation period, the medium was collected and kept at 4°C until extraction. The tissues were rinsed four times with ice-cold Ringer's buffer and homogenized in 750 μl of trichloroacetic acid. Steroids contained in the tissue homogenates and incubation media were extracted three times in 1 ml of dichloromethane as previously described (5). The organic phase containing the steroids was evaporated under nitrogen. The extract was redissolved in a solution consisting of 65% water/trifluoroacetic acid (99.9/0.1, vol/vol; solution A) and 35% methanol/water/trifluoroacetic acid (90/9.98/0.02, vol/vol/v; solution B) and prepurified on Sep-Pak C₁₈ cartridges (Waters) equilibrated with a solution made of 65% solution A and 35% solution B. Steroids were eluted with 4 ml of a solution made of 10% solution A and 90% solution B. The solvent was evaporated in a Speed-Vac Concentrator (Savant), and the steroids contained in the extract were analyzed by HPLC.

HPLC.

Steroids extracted from the tissue homogenates and the incubation media were analyzed by reversed-phase HPLC on a Gilson liquid chromatograph (model 305 master pump, model 306 slave pump) equipped with a manual injector (rheodyne model 7161) and a Nova-Pak C₁₈ column (0.39 × 30 cm; Waters). The HPLC column was equilibrated with a solution made of 60% solution A and 40% solution B (vol/vol), and the Sep-Pak-prepurified extracts were injected at a flow rate of 1 ml/min. The gradient used (40% to 100% solution B over 104 min, with four isocratic steps at 40%, 64%, 80%, and 100% of solution B) is presented in Fig. 4. Tritiated compounds eluted from the HPLC column were directly quantified with a flow scintillation analyzer (Radiomatic Flo-One\Beta A-500; Packard) equipped with a 486DX50 PC computer for measurement of the percentage of total radioactivity contained in each peak. HPLC standards, consisting of synthetic steroids, were detected by UV absorption at 240 nm, using a Gilson UV detector (model 115).

Figure 4.

Analysis of radioactive steroids extracted from the tissue (A and B) or medium (C and D) after a 2-h incubation of frog hypothalamic explants with [³H]pregnenolone in the absence (A and C) or presence (B and D) of 10⁻⁶ M GABA. The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed lines represent the gradient of secondary solvent (% solution B). The arrows indicate the elution position of standard steroids: 17OH-Δ⁵P, 17-hydroxypregnenolone; 5α-DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; F, cortisol; S, 11-deoxycortisol; T, testosterone; 17OH-P, 17-hydroxyprogesterone; P, progesterone; Δ⁵P, pregnenolone.

Quantification of Steroid Biosynthesis and Statistical Analysis.

The amount of radioactive steroids formed by conversion of [³H]Δ⁵P was expressed as a percentage of the total radioactivity contained in all peaks resolved by HPLC, including [³H]Δ⁵P itself. Each value is the mean of four independent experiments. Statistical analysis was performed by ANOVA, followed by a Dunnett's or a Bonferroni's multiple comparison test.

Results

Immunocytochemistry.

As previously reported (5), the rabbit antibody raised against human placental type I 3β-HSD produced intense and specific staining of several neuronal populations in the frog hypothalamus. Labeling of consecutive sections of the frog diencephalon with the antiserum against 3β-HSD and the antiserum against the GABA_A receptor α₃ subunit showed that several 3β-HSD-positive neurons also contained α₃ subunit-like immunoreactivity in the anterior preoptic area (Fig. 1 A and B), the dorsal and ventral hypothalamic nuclei, the nucleus of the periventricular organ, and the posterior tuberculum (Fig. 1 C and D). However, in the hypothalamic nuclei, the GABA_A receptor α₃ subunit-immunofluorescent material was not found in all 3β-HSD-positive neurons (Fig. 1). Reciprocally, 3β-HSD-like immunoreactivity was not detected in all GABA_A receptor α₃ subunit-positive cells (Fig. 1).

Figure 1.

Confocal laser scanning microscope photomicrographs comparing the distribution of 3β-HSD- and GABA_A receptor α₃ subunit-like immunoreactivity in the frog hypothalamus. (A and B) Adjacent frontal sections through the anterior preoptic area are labeled with the antiserum against 3β-HSD (A) or the polyclonal antibodies against the α₃ subunit (B). (C and D) Adjacent frontal sections through the posterior tuberculum labeled with the antiserum against 3β-HSD (C) or the polyclonal antibodies against the α₃ subunit (D). Open arrows, neurons expressing only the 3β-HSD-like immunoreactivity; arrowheads, neurons expressing only the GABA_A receptor α₃ subunit-like immunoreactivity; filled arrows, neurons expressing both 3β-HSD- and GABA_A receptor α₃ subunit-like immunoreactivities. (Bars = 10 μm.)

Double labeling of brain slices with the 3β-HSD antiserum and the monoclonal antibody against the β₂/β₃ subunits of the GABA_A receptor showed that part of the 3β-HSD-positive neurons localized in the posterior tuberculum (Fig. 2 A–C) and the nucleus of the periventricular organ (Fig. 2 D–F) also exhibited β₂/β₃ subunit-like immunoreactivity. The occurrence of β₂/β₃ subunit-immunoreactive material in 3β-HSD-positive neurons was also observed in the anterior preoptic area, the suprachiasmatic nucleus, and the ventral and dorsal hypothalamic nuclei (data not shown). Most neurons that expressed the α₃ subunit also contained the β₂/β₃ subunit. However, a few neurons exhibited only α₃ subunit- or only β₂/β₃ subunit-like immunoreactivity (data not shown).

Figure 2.

Dual-channel confocal laser scanning microscope photomicrographs comparing the distribution of 3β-HSD and GABA_A receptor β₂/β₃ subunit-like immunoreactivity in the frog hypothalamus. (A–C) Frontal section through the posterior tuberculum labeled with the antiserum against 3β-HSD revealed with DAR_S/TXR (A; arrows) or the monoclonal antibody against the β₂/β₃ subunits revealed with GAM_S/Alexa-488 (B; arrowheads). Combination of the two images acquired in A and B revealed the presence of β₂/β₃ subunit-like immunoreactivity in a subset of 3β-HSD-positive neurons (C; arrows). (Bars = 10 μm.) (D–F) Frontal section through the nucleus of the periventricular organ labeled with the antiserum against 3β-HSD revealed with DAR_S/TXR (D) or the monoclonal antibody against the β₂/β₃ subunits revealed with GAM_S/Alexa-488 (E). Combination of the two images acquired in D and E revealed the coexistence of 3β-HSD- and β₂/β₃ subunit-like immunoreactivity in the same neuron (F). (Bars = 2 μm.)

Preincubation of the 3β-HSD antiserum with purified human type I 3β-HSD (10⁻⁶ M) resulted in complete loss of the immunoreaction (Fig. 3 A and B). Similarly, preincubation of the antiserum against the GABA_A receptor α₃ subunit with the synthetic peptide hapten totally abolished immunostaining (Fig. 3 C and D). No fluorescence was observed when the primary antibodies against 3β-HSD, α₃ subunit, or β₂/β₃ subunits were replaced by PBS (data not shown).

Figure 3.

Labeling of consecutive coronal sections through the anterior preoptic area, showing the specificity of the immunocytochemical reactions. (A and B) Specificity control of the 3β-HSD immunostaining. Adjacent sections were incubated with the antibodies against 3β-HSD (A) or with the antibodies preabsorbed with purified human type I 3β-HSD (10⁻⁶ M) (B). (C and D) Specificity control of the α₃ subunit immunostaining. Adjacent sections were incubated with the antibodies against the α₃ subunit (C) or with the antibodies preabsorbed with the purified peptide hapten (10⁻⁶ M) (D). (Bars = 10 μm.)

Effect of GABA on Neurosteroid Biosynthesis.

After a 2-h incubation of frog hypothalamic explants with [³H]Δ⁵P, reversed-phase HPLC analysis of the tissue homogenates and incubation media made it possible to resolve several radioactive compounds, including 17OH-Δ⁵P, 5α-DHT, DHEA, 17OH-P, testosterone, and P (Fig. 4 A and C). In the presence of GABA (10⁻⁶ M), the levels of tritiated metabolites newly synthesized from [³H]Δ⁵P were markedly attenuated in the tissue extracts (Fig. 4B) and the incubation media (Fig. 4D). Exposure of frog hypothalamic slices to graded concentrations of GABA (10⁻⁸ to 10⁻⁴ M) induced a dose-dependent decrease in the biosynthesis of 17OH-Δ⁵P/5α-DHT (Fig. 5A), DHEA (Fig. 5B), 17OH-P (Fig. 5C), and P (Fig. 5D). Incubation of the tissue explants with GABA (10⁻⁶ M) significantly reduced also the amount of ³H-labeled steroids secreted into the incubation medium: 17OH-Δ⁵P/5α-DHT, −45% (P < 0.05); DHEA, −25% (P < 0.05); 17OH-P, −53% (P < 0.01) and P, −35% (P < 0.05).

Figure 5.

Effect of graded concentrations of GABA on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C) and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of GABA. Values are the mean (±SEM) of four independent experiments.

Effect of GABA Receptor Agonists and Antagonists on Neurosteroid Biosynthesis.

The pharmacological characteristics of the receptors involved in the action of GABA were studied using various GABA_A and GABA_B receptor agonists and/or antagonists. Incubation of frog hypothalamic slices with the selective GABA_A receptor agonist muscimol (10⁻⁵ M) mimicked the inhibitory effect of GABA on the conversion of [³H]Δ⁵P into radioactive 17OH-Δ⁵P/5α-DHT, DHEA, 17OH-P, and P (Fig. 6). In contrast, the GABA_B receptor agonist baclofen (10⁻⁵ M) did not significantly modify the level of steroid synthesis by hypothalamic explants (Fig. 6).

Figure 6.

Effects of GABA (10⁻⁵ M), the GABA_A agonist muscimol (10⁻⁵ M), and the GABA_B agonist baclofen (10⁻⁵ M) on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C), and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Each value is the mean (±SEM) of four independent experiments. **, P < 0.01 by one-way ANOVA followed by a post hoc Dunnett's test; NS, not statistically different vs. control.

The inhibitory effect of GABA on the formation of 17OH-Δ⁵P/5α-DHT, DHEA, 17OH-P, and P was completely reversed by the specific GABA_A receptor antagonists bicuculline (10⁻⁵ M) and SR95531 (10⁻⁵ M; Fig. 7). In addition, both bicuculline (10⁻⁵ M) and SR95531 (10⁻⁵ M) induced on their own a modest stimulation of the conversion of [³H]Δ⁵P into 17OH-Δ⁵P/5α-DHT, DHEA, 17OH-P, and P (Fig. 7).

Figure 7.

Effects of GABA (10⁻⁶ M) in the absence or presence of the GABA_A antagonists SR95531 (10⁻⁵ M) and bicuculline (10⁻⁵ M) on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C), and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Each value is the mean (±SEM) of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA followed by a post hoc Bonferroni's test; NS, not statistically different.

Discussion

Most of the central effects of neurosteroids are mediated through allosteric modulation of GABA_A receptors, but a direct effect of GABA on neurosteroid biosynthesis has never been demonstrated. The present study provides evidence for the expression of GABA_A receptors in neurosteroid-secreting neurons. Our data also demonstrate that GABA, acting through GABA_A receptors, exerts an inhibitory effect on the production of Δ⁵-3β-hydroxysteroid and Δ⁴-3-ketosteroids in the frog hypothalamus.

Among the 15 different GABA_A receptor subunits that have been characterized to date (19–21), the α₁ and β₂ subunits are, by far, the most common subunits occurring in native GABA_A receptors in the brain (22–25). The α₃ subunit is also contained in a significant proportion of GABA_A receptors (22, 24, 25). To investigate whether 3β-HSD-containing neurons also express GABA_A receptors, we have used a specific polyclonal antibody against the α₃ subunit (17, 18, 26) and a monoclonal antibody against the β₂/β₃ subunits that also cross-reacts with the α₁ subunit (24, 27, 28). In a recent report, this latter antibody has been used successfully to localize the GABA_A receptor complex in the brain of the frog Rana pipiens (29). The present study reveals that most of the 3β-HSD-positive neurons located in the anterior preoptic area, the suprachiasmatic nucleus, the posterior tuberculum, the nucleus of the periventricular organ, and the dorsal and ventral hypothalamic nuclei also contain GABA_A receptor α₃ and β₂/β₃ subunit-like immunoreactivity. Because recombinant GABA_A receptors consisting of α₁/α₃, β₂/β₃, and γ₂ subunits are functionally activated by GABA (22, 30, 31) and exhibit high affinity for central-type benzodiazepines (28), our data suggested that GABA could actually modulate the biosynthesis of steroids in 3β-HSD-containing neurons. Incubation of frog hypothalamic explants with graded concentrations of GABA provoked a dose-dependent inhibition of the conversion of [³H]Δ⁵P into [³H]Δ⁴-3-ketosteroids such as P, 17OH-P, and 5α-DHT. GABA also inhibited the formation of [³H]Δ⁵-3β-hydroxysteroids, including 17OH-Δ⁵P and DHEA. These observations indicate that, in the frog brain, GABA inhibits the biological activity of at least two steroidogenic enzymes, i.e., 3β-HSD and 17α-hydroxylase. Consistent with this notion, in vivo studies have recently shown that depression of GABA neurotransmission by the GABA synthesis inhibitor isoniazid increases the content of Δ⁵P, P, and THDOC (tetrahydrodeoxycorticosterone) in the rat brain (14). The fact that GABA also significantly inhibited the concentration of newly synthesized steroids in the incubation medium indicates that GABA simultaneously reduces biosynthesis and release of neurosteroids by hypothalamic neurons.

The present report demonstrates that the action of GABA on neurosteroidogenesis is mediated through GABA_A receptors: (i) The selective GABA_A receptor agonist muscimol mimicked the inhibitory effect of GABA on the biosynthesis of 17OH-Δ⁵P/5α-DHT, P, 17OH-P, and DHEA, whereas the GABA_B receptor agonist baclofen did not affect neurosteroid synthesis. (ii) The effect of GABA was abolished by the GABA_A receptor antagonists bicuculline and SR95531. We have also noticed that bicuculline and SR95531 enhanced the spontaneous production of 17OH-Δ⁵P/5α-DHT, DHEA, 17OH-P, and P, suggesting that endogenous GABA, acting on GABA_A receptors, actually exerts a tonic inhibitory control on hypothalamic neurons that synthesize neurosteroids. The effect of GABA on pregnenolone biosynthesis in the rat retina is also mediated through GABA_A receptors, although, in this latter model, GABA exerts a stimulatory effect on steroid production (13).

In conclusion, the present study provides evidence for the occurrence of GABA_A receptors on 3β-HSD-immunoreactive neurons in the brain of vertebrates. This report also demonstrates that GABA, acting through GABA_A receptors, reduces the rate of conversion [³H]Δ⁵P into [³H]Δ⁴-3-ketosteroids and [³H]Δ⁵-3β-hydroxysteroids. Taken together, these observations indicate that GABA exerts a direct inhibitory effect on steroid-producing neurons. Because neurosteroids are potent regulators of GABA_A receptor function (10, 11, 32), our data suggest the existence of an ultrashort regulatory loop by which neurosteroids may regulate their own production through the modulation of GABA_A receptor activity.

Abbreviations

P

progesterone

Δ⁵P

pregnenolone

DHEA

dehydroepiandrosterone

GABA

γ-aminobutyric acid

3β-HSD

3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase

5α-DHT

dihydrotestosterone

17OH-Δ⁵P

17-hydroxypregnenolone

17OH-P

17-hydroxyprogesterone

GAM_S/Alexa-488

Alexa-488-conjugated goat anti-mouse γ-globulins

DAR_S/TXR

Texas Red-conjugated donkey anti-rabbit γ-globulins