An immunoblot analysis was performed as described previously (Yamaguchi et al., 2007 (link)). In brief, the telencephalons from 0- and 5-day-old chicks reared in the dark were dissected after anesthesia. For the detection of GABA-A receptors, an anti-GABA-A receptor subunit alpha 1 rabbit polyclonal antibody was used as the primary antibody (ab33299, 1:1,500; Abcam plc, Cambridge, United Kingdom), while an anti-rabbit horseradish peroxidase-conjugated antibody (1:1,000; GE Healthcare, Chicago, IL, United States) was used as the secondary antibody. To detect GABA-B receptors, an anti-GABA-B receptor subunit 2 rabbit monoclonal antibody (ab75838, 1:1,500; Abcam plc) was used, while an anti-rabbit horseradish peroxidase-conjugated antibody (1:1,000, GE Healthcare) was used as the secondary antibody. Data of each sample were normalized to the expression of beta-actin as detected by an anti-beta-actin mouse monoclonal antibody (A5316, 1:1,000, Sigma-Aldrich Co.). The band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, United States), and the ratios of the band intensities were calculated.
Ab33299
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab33299 is a lab equipment product. It is designed for use in various scientific research applications. The core function of this product is to provide a tool for researchers to perform their experiments.
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Lab products found in correlation
Ab55051, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by GE Healthcare (1 mentions)
A5316, by Merck Group (1 mentions)
Ab75838, by Abcam (1 mentions)
Alexa fluor 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Ab32454, by Abcam (1 mentions)
Ab193311, by Abcam (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab11427, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab65783, by Abcam (1 mentions)
Supersignal west femto chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit immunoglobulin g, by Thermo Fisher Scientific (1 mentions)
Ab133265, by Abcam (1 mentions)
Ab8891, by Abcam (1 mentions)
Ab2064, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Sc 293072, by Santa Cruz Biotechnology (1 mentions)
9 protocols using ab33299
1
GABA Receptor Expression in Chick Telencephalon
2
Immunohistochemical Analysis of Hippocampal and Amygdalar Neurons
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Brains (n = 6/treatment group from different litters generated in Experiment 2) were processed for immunohistochemistry. Coronal cryostat sections (12 μm) including the hippocampus (CA1, CA2, CA3) and the basolateral amygdala were mounted as contiguous triplicates. Sections were fixed in cold methanol (−20 °C), blocked with 5% goat serum, 0.3% Triton X-100 in phosphate buffered saline (PBS) for 2h at 4 °C, followed by an overnight incubation at 4 °C in primary antibody against either parvalbumin (1:500, ab11427; abcam), MAP2 (1:500, ab32454; abcam), GABA Aα1 (1:500, ab33299; abcam), GABA Aα2 (1:500, ab193311; abcam) or GABA B1 (1:500, ab55051; abcam) in PBS with 1% bovine serum albumin (BSA), 0.3% Triton X-100. Sections were incubated with Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 568 anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) at 1:500 for 2h at 4 °C. Vectashield Mounting Medium with DAPI (Vector Laboratories, USA) was used to mount coverslips.
3
Immunodetection of GABAA Receptor Subunits
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The following antibodies were used for immunoprecipitation (IP), western blot (WB), or immunocytochemistry (ICC): Gabra1 (mouse, NeuroMab clone N95/35, IP), Gabra1 (rabbit, Abcam ab33299, WB 1:1000, ICC 1:1000), Gabra4 (mouse, NeuroMab clone N398A/34, IP, ICC 1:500), Gabra4 (rabbit, PhosphoSolutions 845A-GA4C, WB 1:1000), Gabrb3 (rabbit, PhosphoSolutions 863A-GB3C, WB 1:1000), p-S408/9 (PhosphoSolutions p1130-4089, WB 1:1000), Sptan1 (rabbit, Cell Signaling 2122S, WB 1:1000), Sptbn1 (rabbit, Abcam ab72239, WB 1:1000), Sptbn2 (rabbit, Proteintech 55107-1-AP, WB 1:1000), Erc2 (rabbit, Abcam ab170862, WB 1:1000), Kcc2 (rabbit, Millipore 07-432, WB 1:2000), Gephyrin (rabbit, Cell Signaling 14304S, WB 1:1000), N-Cadherin (mouse, Cell Signaling 14215S, WB 1:1000), Calreticulin (rabbit, Cell Signaling 12238S, WB 1:1000, ICC 1:500), Hsp60 (rabbit, Cell Signaling 12165S, WB 1:1000), Hsp90 (rabbit, Cell Signaling 4877S, WB 1:1000), GAPDH (mouse, Santa Cruz sc-32233, WB 1:5000), Goat anti-mouse Alexa Fluor 488 (Thermo Fisher A11029, ICC 1:1000), Goat anti-rabbit Alexa Fluor 568 (Thermo Fisher A11011, ICC 1:1000), Donkey anti-mouse conjugated HRP (Jackson ImmunoResearch 715-035-150, WB 1:5000-7000), Donkey anti-rabbit conjugated HRP (Jackson ImmunoResearch 711-035-152, WB 1:2500-7000).
4
Immunohistochemical Analysis of GABA and NMDAR2B
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Detailed steps as described in our previous articles (Wu et al., 2010 (link); Zhou et al., 2017 (link)). The cryosections were then incubated with rabbit polyclonal antibody against GABAA alpha 1 (ab33299, 1:100; Abcam, Cambridge, MA, USA), rabbit polyclonal antibody against NMDAR2B (ab65783, 1:200; Abcam, Cambridge, MA, USA). The secondary antibodies (all from Invitrogen-Molecular Probes, Eugene, OR, USA; Zhou et al., 2017 (link)) were 555-conjugated donkey anti-rabbit IgG antibody (A31572, 1:1000) and 488-conjugated goat anti-rabbit IgG antibody (A11070, 1:500).
5
Western Blot Analysis of Synaptic Receptors
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Detailed steps as described in our previous articles (Wu et al., 2010 (link); Zhou et al., 2017 (link)). The primary antibodies: rabbit polyclonal antibody against GABAA alpha 1 (ab33299, 1:1000; Abcam, Cambridge, MA, USA), rabbit monoclonal antibody against NMDAR2B (4212S, 1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal antibody against NMDAR1 (#5704, 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal antibody against NMDAR2A (ab133265, 1:1000; Abcam, Cambridge, MA, USA). The membranes were incubated with goat anti-rabbit immunoglobulin G (31460, 1:8000; Thermo Fisher, West Grove, PA, USA). Then, the immunoreactivity was visualized using the SuperSignal West Femto Chemiluminescent Substrate kit (34094, Thermo Fisher Scientific). The relative intensities of the protein bands using ImageJ software (Zong et al., 2017 (link)). α-Tubulin and GAPDH were used as the internal standard.
6
Immunohistochemical Analysis of GABAergic and Inflammatory Markers
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Paraffin sections were baked overnight at 65°C. Deparaffinization and hydration were performed in xylene and graded ethanol to distilled water. Antigen retrieval was performed in tris-ethylenediamine tetraacetic acid (EDTA) buffer in a microwave oven and cooled to room temperature. After washing, the endogenous peroxidase was removed using 0.3% H2O2, and the sections were subjected to blocking for 15 min. Then, the sections were incubated with primary antibody at room temperature for 2 h. The antibodies used were: anti-GABAAR α1-6 (sc-376282, Santa Cruz, California, USA, 1:50); anti-GABAAR α1 (ab33299, Abcam, Cambridge, UK, 1:100); anti-GABA (ab8891, Abcam, 1:200); anti-TLR4 (sc-293072, Santa Cruz, 1:100); anti-MyD88 (ab2064, Abcam, 1:200); anti-NF-κB p65 (ab86299, Abcam, 1:200). After washing, the tissues were labeled with secondary antibodies for 1 h and developed with 3,3′-diaminobenzidine, followed by washing with tap water. Counterstaining was performed using hematoxylin, and the slides were rinsed and mounted with dibutylphthalate polystyrene xylene mounting medium. Finally, the sections were observed under a microscope and the results were measured with Image J.
7
Western Blot Analysis of Retinal Proteins
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Protein preparation and Western blotting were performed according to previously described methods (Zhou et al., 2017a (link)) with modifications. Briefly, retinal lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. In total, 20 μg of each sample was separated by SDS-PAGE and electrotransferred onto PVDF membranes (Immobilon-P; Millipore). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: GABA-alpha receptor (GABAAR; ab33299, rabbit, 1:500; Abcam), protein kinase C-alpha (PKC-α; ab32122, rabbit, 1:1,000; Abcam), and B-cell lymphoma-2 (rabbit, 28 kDa, 1:1,000, Beyotime Institute of Biotechnology, China). The membranes were then incubated with horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1:5,000; Cell Signaling Technology). The relative intensities of the protein bands were quantified by scanning densitometry using ImageJ software. Mouse monoclonal antibodies against β-tubulin (ab78078, 1:2000; Abcam) and β-actin (mouse, 43 kDa, A5441, 1:5,000, Sigma-Aldrich, USA) were used as internal standards.
8
Glutamate Receptor Inhibition in Cortical Cultures
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Mixed, astrocyte-only and neuron-only cortical cultures were prepared from dissociated rat E18 cortical tissue [29 (link)]. The effect of glutamate receptor inhibition was investigated by adding 10 μM MK801 to the cultures 4 h before incubation with conditioned media. Immunocytochemical assessment was performed as described previously [28 (link)] using antibodies against MAP2 (Synaptic Systems 188004; 1:2000), GFAP (CST 3670; 1:1000), tyrosine hydroxylase (abcam ab112; 1:500), GluN1 (Millipore Ab9864; 1:200), GluN3α (Millipore 07-356; 1:200), GABA Aα1 (abcam ab33299; 1:250) and GABA B1 (abcam ab55051; 1:250).
9
Immunohistochemical Analysis of Hippocampal Interneurons
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Brains were fixed by transcardial perfusion (4% paraformaldehyde) followed by post-fixation (1 h at 4 °C), and vibratome sections (50 μm thickness) were prepared. Serial sections at the level of the dorsal hippocampus were incubated overnight with the following antibodies; anti-parvalbumin (PV) mouse monoclonal (P3088, Sigma-Aldrich, USA; 1:2000 dilution); anti-somatostatin (SOM) rabbit polyclonal (T-4102, Bachem, UK; 1:2000 dilution); anti-GABAA receptor α1 subunit (GABRA1) rabbit polyclonal (ab33299, Abcam, 1:1000 dilution); anti-GABAA receptor α3/GABRA3) rabbit polyclonal (AB5594, Millipore, 1:1000 dilution); and anti-GABAA receptor α5 (GABRA5) rabbit polyclonal (ab 10098, Abcam, 1:1000 dilution). Biotin-conjugated secondary antibody and streptavidin conjugated to appropriate fluorophores (AlexaFluor 488/594, Invitrogen Life Technologies, USA) were used to reveal the signal.
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