After deparaffinization and hydration, tissue sections were boiled for 15 min in sodium citrate buffer, pH 6.0 to retrieve the epitope. Endogenous peroxidase was blocked by incubation of brain sections with 1% hydrogen peroxide for 15 min. Immunostaining was performed with ImmPress Reagent kit (Vector laboratories) according to manufacturer's instructions. Following primary antibodies were used: anti-TCTP (Abcam, ab37506, 1:1000), anti-MAP2a+2b+2c (Abcam, ab68852, 1:100), anti-NMDAR2B (Abcam, ab93610, 1:1000), and anti-synapsin II (Sigma Aldrich, S2822, 1:1000). Signals reacted with primary antibodies were substituted with normal rabbit IgG (Abcam, ab27478). Secondary antibody was reacted with ABC kit and 0.05% DAB solution (Sigma-Aldrich) was used as a chromogen. After staining with cell nuclei using Harris hematoxylin (Sigma-Aldrich), the sections were mounted with “Permount” (Fisher Scientific). Stained sections were analyzed under the microscope (Axio Scope. A1, Zeiss), and representative photographs were made with a digital camera.
Ab37506
Manufactured by Abcam
Sourced in United Kingdom
Ab37506 is a laboratory equipment product. It serves a core function related to the product's intended use, but no further details or interpretation are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab27478, by Abcam (1 mentions)
Immpress reagent kit, by Vector Laboratories (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
Abc kit, by Merck Group (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
Yap taz, by Cell Signaling Technology (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Alexa fluor 488 or alexa fluor 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Ptctp, by Cell Signaling Technology (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Cxcl10, by BD (1 mentions)
Ab97959, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp ab6721, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Ab195977, by Abcam (1 mentions)
8 protocols using ab37506
1
Immunohistochemical profiling of neuronal markers
2
Protein Expression Analysis Protocol
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Anti-TCTP antibodies AB37506 (polyclonal; for immunohistochemistry), and AB58362 (monoclonal; for western blotting) were purchased from Abcam (UK) via Sapphire Bioscience, Waterloo, NSW Australia. Phospho-ribosomal protein S6 (Ser 240/244; #2215), and secondary HRP-linked antibodies were from Cell Signalling Technology, purchased via Genesearch, Arundel, QLD Australia. Anti-α-and β-tubulin antibodies were from Amersham Biosciences (UK).
3
Quantifying TAZ and HRF in Samples
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Protein samples were collected from cultured cells and tissues (spleen, lymph nodes, and back skin) and extracted using lysis buffer. Serum samples were also collected from the blood of WT and TAZ KO mice and pre-treated to remove serum albumin proteins. Protein extracts and serum samples were analyzed by SDS-PAGE and electrotransfer, followed by incubation with antibodies against HRF (AB37506, Abcam) and TAZ (#8418, YAP/TAZ, Cell signaling technology). A similar amount of protein loading was confirmed by immunoblotting of β-actin (BS-0061R, Bioss, Woburn, MA, USA) and by staining Ponceau S (Sigma). The protein band signal was quantitated using Image J software.
4
Histological Analysis of Skin and Lymph Nodes in TAZ KO Mice
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The dorsal skin, ear skin, and lymph nodes were collected from WT and TAZ KO mice. The tissues were fixed, embedded, and sectioned at 4 μm thickness. Skin section slides were stained with either hematoxylin and eosin (H&E), trichrome, or toluidine blue, followed by microscopic observation. For immunohistochemistry, skin and lymph node sections were fixed and incubated with antibodies against HRF (ab37506, Abcam), followed by incubation with DAPI (1 μg/mL). Images were viewed under a fluorescence microscope (Axio Observer 7, Carl Zeiss Jena, Germany) at the Ewha Drug Development Research Core Center.
5
Localization of LAMP1, TAZ, and HRF
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BMMCs were treated with or without LPS (1 µg/mL) for 24 h and treated with either vehicle or C48/80 (50 µg/mL) for 5 min before harvest. The cells were fixed with 4% paraformaldehyde and incubated with antibodies against LAMP1 (sc-19992, Santa Cruz Biotec), TAZ (#72804, Cell signaling technology, Danvers, MA, USA; or #560235, BD Bioscience), and HRF (AB37506, Abcam, Cambridge, MA, USA), followed by staining with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies (Invitrogen). Cell nuclei were stained with DAPI (1 µg/mL). Cells were observed using a confocal laser scanning microscope (ZEISS LSM 880 with Airyscan, Oberkochen, Germany).
6
Quantitative Western Blot Profiling
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Lysate extracted from a total of 5 × 105 cells was used to perform western blots. Primary antibodies against TCTP (ab37506, Abcam), pTCTP(#5251, Cell signaling), pEGFR(Life technologies, 44784G), EGFR(#4267, Cell signaling), PLK1 (#4535, Cell signaling), MCL-1 (sc-819, Santa Cruz Biotechnology), CXCL10 (551215, BD Biosicences), β-actin (M177-3, MBL) were used. Western blotting was followed by incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase, anti-rabbit IgG-HRP (Enzo, Cat ADI-SAB-300-J), and anti-mouse IgG-HRP (Enzo, Cat ADI-SAB-100-J). The immunoreactive bands were developed with the chemiluminescence ECL Detection system (GE Healthcare), and signals were detected using a luminescent image analyzer (LAS-4000 Mini).
7
Immunohistochemical Analysis of Tissue Samples
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For immunohistochemistry, the dissected tissues were initially fixed in 10% formalin solution and processed to get paraffin-embedded tissue. The microtome was set at 6 µm, and the sections obtained were fixed on a microscopic glass slide. Following deparaffinization with xylene, they were hydrated with water. To block the endogenous peroxidase activity, the sections were immersed in freshly prepared 10% H2O2 and 10% methanol in 1X phosphate-buffered saline (PBS) for 20 minutes. After washing with 1XPBS, the sections were treated with 0.1% trypsin in 0.1% CaCl2 at 37°C for 5 minutes. In order to facilitate binding of specific antibodies, the sections were incubated with the primary antibody overnight at 4°C followed by the addition of a suitable secondary antibody for 30 minutes at room temperature. After washing the tissue sections, the slides were developed using a DAB (diaminobenzidine) kit as a chromogen for detecting signals. For immunohistochemistry experiments, the following primary antibodies were used: anti-Sox2 antibody (ab97959) and anti-TCTP antibody (ab37506) from Abcam (Cambridge, UK). Similarly, the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab6721) from Abcam was used to detect the signals.
8
Western Blot Analysis of Protein Expression
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Late third instar larvae or S2 cells were lysed with lysis buffer (20 mM HEPES pH7.4, 70 mM KCl, 10 mM EDTA, 10 mM EGTA, 2 mM DTT, 1 mM PMSF, 0.1% Igepal CA-630, 16% Glycerol, and Roche EDTA-free Protease inhibitor cocktail) chilled on ice. The lysates were boiled with sample loading buffer at 95 °C for 10 mins. The total protein samples were loaded on 7.5% or 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking at RT, membranes were probed sequentially in primary antibody solution and HRP-conjugated secondary antibody solution diluted with 2% dry milk or 2% BSA in TBST (140 mM NaCl; 3 mM KCl; 25 mM Tris pH7.4; 0.1% Tween 20). Pierce ECL western blotting substrate (Thermo) was used to detect immunostained proteins. A cytosolic fraction from salivary glands was isolated as described [35 (link)]. Primary antibodies were rabbit anti-hFOXO1 (1:1000; A2934, Abclonal), rabbit anti-dFoxo (1:1000; ab195977, Abcam), rabbit anti-hTCTP (1:2000; ab37506, Abcam), rabbit anti-Tctp (1:2500) [18 (link)], mouse anti-βTub (1:5000; E7, DSHB), and rabbit anti-GFP (1:10000; ab290, Abcam).
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