The following main reagents were used: Aβ25–35 fragment (Sigma, United States), CEF (Roche, Switzerland), a mouse monoclonal antibody to Aβ (cat. # NBP2-13075, 1:1,000 dilution; Novus Biologicals, United States), a rat monoclonal antibody to CD54/ICAM-1 (cat. # 16-0542-81, 1:300 dilution; eBioscience, Thermo Fisher Scientific, United States), a goat polyclonal antibody to microglial marker AIF-1/IBA1 (cat. # NB100-1028, 1:200 dilution; Novus Biologicals, United States), an Alexa Fluor 568-conjugated goat anti-mouse IgG polyclonal antibody (cat. # ab175473, 1:400 dilution, Abcam, United Kingdom), an Alexa Fluor 594-conjugated goat anti-rat IgG polyclonal antibody (cat. # ab150160, 1:500 dilution; Abcam, United Kingdom), and an Alexa Fluor 488-conjugated donkey anti-goat IgG polyclonal antibody (cat. # ab150129, 1:200 dilution; Abcam, United Kingdom).
Nbp2 13075
Manufactured by Novus Biologicals
Sourced in United States
NBP2-13075 is a primary antibody that recognizes the GAPDH protein. GAPDH is a ubiquitously expressed enzyme involved in glycolysis. This antibody can be used in various immunoassay applications to detect and quantify GAPDH expression.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated donkey anti rabbit igg, by GE Healthcare (2 mentions)
Nbp2 25093, by Novus Biologicals (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab150160, by Abcam (1 mentions)
Aβ25 35 fragment, by Merck Group (1 mentions)
Nb100 1028, by Novus Biologicals (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ox ldl, by Biomedical Technologies (1 mentions)
Microtiter plate reader, by Molecular Devices (1 mentions)
Ab11267, by Abcam (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab15456, by Abcam (1 mentions)
Ab3347, by Abcam (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab201061, by Abcam (1 mentions)
Mabs1158 for wb, by Merck Group (1 mentions)
Hpa017336, by Merck Group (1 mentions)
A10438, by ABclonal (1 mentions)
7 protocols using nbp2 13075
1
Immunofluorescence Localization of Aβ, ICAM-1, and Iba1
2
Amyloid-like Structure Detection on Immobilized Proteins
3
Immunohistochemical Analysis of Mouse Brain Tissue
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3 μm-thick paraffin coronal sections of the mouse brain tissue were used for immunohistochemical analysis. Immunohistochemistry was performed according to the manufacturer’s instructions of the PV-6000D kit (Zhongshanjinqiao, Beijing, China). In detail, paraffin sections were baked at 65°C for 120 min, deparaffinized in xylene, and rehydrated through graded alcohol. Antigen retrieval was performed by microwave heating at 99°C in citrate buffer for 5 min three times and endogenous peroxidase was blunted by incubating samples with 3% H2O2 for 10 min. The sections were then placed in blocking buffer (10% natural goat serum) for 30 min at 37°C. The sections were incubated overnight at 4°C with the following primary antibodies: beta Amyloid antibody (MOAB-2) (1:1,000, NBP2-13075, Novus Biologicals), GFAP (E4L7M) XP® Rabbit mAb (1:200, #80788, Cell Signaling Technology), and Iba1/AIF-1 (E4O4W) XP® Rabbit mAb (1:2000, #17198, Cell Signaling Technology). After that, secondary antibody working solution was incubated at 37°C for 1 h. Finally, the slides were then dehydrated in ethanol followed by xylene and sealed with cover slips. The percent area of positive labeling was analyzed at ×20 magnification.
4
Immunohistochemical Analysis of Amyloid-Beta
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Before immunohistochemistry, brain slices were blocked with hydrogen peroxide solution at room temperature for 20 min. Afterward, slices were incubated in in antibody against Aβ (#NBP2-13075, Novus, Littleton, CO, USA) at 4 °C overnight and horseradish peroxidase labeled second antibody at room temperature for 1 h. The chromogenic reaction was terminated by washing sections in PBS until slices’ fully stained. Stained tissue sections were then dehydrated in alcohol and counter-stained with hematoxylin. After differentiation, slices were promptly washed in PBS for 3 times and analyzed by ImageJ Software.
5
Detecting Amyloid-like Structures on Immobilized IgG
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The presence of amyloid-like structures on immobilized IgG was detected as described previously (5 (link)). Microtiter wells (Corning, 9018) were coated with 10 μg/ml of monomeric IgG (mono-IgG) or agg-IgG in TBS, pH 7.2, and incubated at 4°C overnight. The unreacted sites in the wells were blocked with TBS containing 0.5% gelatin at room temperature for 45 min. Both, polyclonal antibodies (Novus, NBP2-25093) and monoclonal antibodies (Novus, NBP2-13075) to amyloid-β peptide 1-42 (Aβ) were used to detect the amyloid-like structures formed on the mono-IgG and agg-IgG following their immobilization. Normal rabbit IgG and normal mouse IgG were used as controls for the antibodies. The antibodies (10 μg/ml) diluted in TBS containing 0.1% gelatin and 0.02% Tween 20 were added to the wells and incubated at 37°C for 1 h. After washing the wells, bound polyclonal anti-Aβ antibodies were detected by using HRP-conjugated donkey anti-rabbit IgG (GE Healthcare) and bound monoclonal anti-Aβ antibodies were detected by using HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific). Color was developed with ABTS reagent and the OD was read at 405 nm in a microtiter plate reader (Molecular Devices). Immobilized Aβ peptide 1-42 (Bachem) was used as a control for immobilized IgG and for anti-Aβ antibodies.
6
Antibody Panel for Alzheimer's Markers
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Antibodies against PTPRT (MABS1158 for WB and HPA017336 for IHC) and Flag (F3165) were from Sigma; pSTAT3 (Y705) (AP0070), ADAM10 (A10438) and ADAM17 ( A0821) antibodies were from ABclonal; antibodies against A42 (ab201061), STAT3 (ab68153), -tubulin (ab7291), β-actin (ab8227), Presenlin-1 (ab15456), VAMP2 (ab3347) and Map2 (ab32454 and ab11267) were obtained from Abcam; antibodies against cleaved-Caspase3 (#9661) , a-tubulin: (#3873S), histon3 (#9715) were purchased from Cell Signaling Tech; antibody against the A plaque 6E10 (SIG-393200) antibody were from Covance; antibody against oligomeric A MOAB-2 (NBP2-13075) and biotin-labeled MOAB-2 (NBP2-13075B) from Novus Biologicals. The secondary antibodies used for immunocytochemistry were as follows: chicken anti-mouse or rabbit Alexa 488; donkey anti-mouse or rabbit Alexa 594 (Invitrogen, Eugene, OR); all were used at a dilution of 1:500. ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, P36962) was used as a nuclear counterstain at 1 µg/ml. DAPT (γ-secretase inhibitor, D5942), Thioflavine S (T1892), Anti-Digoxigenin-AP (Fab fragments, 11093274910), and Phorbol 12myristate 13-acetate (PMA, p8139) were ordered from Sigma-Aldrich.
7
Optimized Immunofluorescence Imaging of Cleared Brain Tissues
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After acquiring the whole brain images from CX3CR1-eGFP, 5xFAD, C57BL/6J mice the brains were further optimized for cryopreservation and sectioning. The course of tissue clearing and imaging in BABB makes the tissue brittle and hard for further processing, Thus, to solve this hurdle we re-hydrated the samples with respective clearing solutions to be able to process samples for cryosectioning. Thereafter, samples were washed with X-100 in PBS for 15 min, blocked for 1 hour at room temperature with 10% serum in PBST. Then incubation with primary antibodies Iba1 (1:1000, Wako, 019-19741), Stathmin 1 (1:300, Novus, NBP1-76798), Neurocan (1:300, abcam, ab31979), S100a11 (1:300, R&D, MAB5167), Thop1 (1:300, Novus, NB400-146), MOAB2 (1:1000, Novus, NBP2-13075), at 4°C for overnight and Alexa conjugated secondary antibodies (1:1000, Goat anti-rabbit IgG Alexa fluor 647, Invitrogen, A21245;
Goat anti-Mouse IgG Alexa Fluor 488, A11029; Goat anti-Mouse IgG Alexa Fluor 594, A11032; Goat anti-Rat IgG Alexa Fluor 594, A11007; Goat anti-Rat IgG Alexa Fluor 488, A11006; ) were incubated for 1 hour at room temperature. Slices were mounted after being stained with Hoechst 33342 (Invitrogen). Images were acquired with 10x, 40x, and 63x objective of confocal microscope (ZEISS LSM880).
Goat anti-Mouse IgG Alexa Fluor 488, A11029; Goat anti-Mouse IgG Alexa Fluor 594, A11032; Goat anti-Rat IgG Alexa Fluor 594, A11007; Goat anti-Rat IgG Alexa Fluor 488, A11006; ) were incubated for 1 hour at room temperature. Slices were mounted after being stained with Hoechst 33342 (Invitrogen). Images were acquired with 10x, 40x, and 63x objective of confocal microscope (ZEISS LSM880).
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