Mouse cerebral cortex tissues were extracted and homogenized in a lysis buffer containing a mixture of protease inhibitors. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected. A rapid Gold BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) was used to determine protein concentrations. The same amount of protein was isolated using 10% SDS-PAGE and then transferred to a PVDF membrane (Billerica Millipore, MA, USA). Anti-CCN1 (1:2000, Proteintech, Beijing, China), anti-Aβ1–42 (1:2000, ab201060, Abcam, USA), anti-IL-1β, anti-TNF-a (1:2000, ab201060, Abcam, USA), and anti-AQP4 (1:2000, Proteintech, Beijing, China) were also used, and anti-cleaved caspase-3 (1:1000, Affinity, China) and anti-p-ERK1/2 (1:2000, Ap0472, ABclonal, Wuhan, China) were cultured overnight at 4 °C. Protein bands were detected using the enhanced chemiluminescence detection system (Cell Signaling Technologies, Beverly, MA, USA), and the ImageJ image analysis program (NIH ImageJ, USA) was used to determine the strength of the immune response bands.
Ab201060
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab201060 is a laboratory equipment product. It is a core component used in various scientific experiments and analyses. The product's primary function is to facilitate specific research tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ba0056, by Boster Bio (4 mentions)
Pb0517, by Boster Bio (4 mentions)
Ab8805, by Abcam (3 mentions)
Ab192890, by Abcam (2 mentions)
Ab191606, by Abcam (2 mentions)
Gb12001, by Wuhan Servicebio Technology (2 mentions)
Ab207612, by Abcam (2 mentions)
Ab9969, by Abcam (2 mentions)
Ab9722, by Abcam (2 mentions)
Ab81283, by Abcam (2 mentions)
P p38 mapk, by Cell Signaling Technology (2 mentions)
Ab76083, by Abcam (2 mentions)
Mab1059, by Merck Group (2 mentions)
Frozen slicer, by Leica (2 mentions)
Bm3894, by Boster Bio (2 mentions)
Zen 2, by Olympus (2 mentions)
Ab150073, by Abcam (2 mentions)
Ab37439 100, by MultiSciences Biotech (2 mentions)
Ab32391, by Abcam (2 mentions)
Ab75814, by Abcam (2 mentions)
26 protocols using ab201060
1
Cerebral Cortex Protein Expression Analysis
2
Western Blotting Analysis of Hippocampal Proteins
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After the behavioral experiment, three mice were randomly selected from each group for Western blotting. The brain tissue of the mice was quickly removed and the hippocampus tissue was separated at low temperature. The treated brain tissue requires extraction of histones at low temperature, followed by grinding and centrifugation of the sample. The protein concentration of the sample is determined from the standard curve and then heated to denature the proteins of the sample. Electrophoresis and membrane transfer are then performed. We use the first antibody to react with the PVDF membrane overnight, put down the second antibody the next day and finally rinse with PBS solution. The gels were then scanned using an automated gel imaging system and analysed for calculation using ImageJ software. Main antibodies and dilution ratio: Anti-Akt (ab81283, Abcam, 1:1000); Anti-beta Amyoid 1-42 (ab201060, Abcam, 1:1000); Anti-IL-1beta (ab9722, Abcam, 1:800); Anti-IL-10 (ab9969, Abcam, 1:800); Anti-PI3K (ab191606, Abcam, 1:1000); Anti-GFAP (BA0056, BOSTER, 1:800); Anti-IBA-1 (PB0517, BOSTER, 1:800); Anti-Beclin1 (ab207612, Abcam, 1:1000); Anti-LC3 (ab192890, Abcam, 1:1000); Anti-p-Akt (ab8805, Abcam, 1:1000); Anti-β-actin (GB12001, Servicebio, 1:2000).
3
Protein Expression Analysis in Brain Tissue
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Brain tissues and cells were lysed for total protein extraction using SDS buffer. Protein concentrations were determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s protocol. Equal amounts of protein were subjected to Western blotting analysis. Antibodies included those specific for Aβ (ab201060, Abcam, Cambridge Science Park, Cambridge, UK), PEDF (MAB1059, Millipore, Boston, MA), PS1 (ab76083, Abcam), phosphorylated c-Jun N-terminal kinase (P-JNK; #3270S, Cell Signaling Technology, Boston, MA), JNK (ab119944, Abcam), BACE1 (#5606S, Cell Signaling Technology), APP (#2452S, Cell Signaling Technology), low-density lipoprotein receptor-related protein 6 (LRP6; SC-25317, Santa Cruz, CA), phosphorylated extracellular signal-regulated kinase (P-ERK; SC-7383, Santa Cruz), ERK (SC-94, Santa Cruz), p38 mitogen-activated protein kinase (MAPK; #9211S, Cell Signaling Technology), P-p38 MAPK (#9212S, Cell Signaling Technology), and β-actin (A544, 2 ml, Sigma).
4
Immunohistochemical Detection of Chlamydia and Amyloid-β
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Immunohistochemistry was performed as previously described27 (link),43 (link),44 (link). Specimens were incubated with goat anti-C. trachomatis/C. pneumoniae (this antibody is used to detect both of these Chlamydia species; Abcam ab20929; 1:400) and/or rabbit anti-Aβ peptide (Abcam ab201060,1:500). Secondary antibodies were donkey anti-goat Alexa Fluor 488 (Abcam ab150129 1:400), donkey anti-rabbit 647 (Invitrogen A31573; 1:500). Antibodies were diluted in blocking buffer (2% bovine serum albumin with 0.3% Triton X-100 in PBS). Cryostat sections were first incubated with blocking buffer for 60 min at room temperature, followed by overnight incubation with primary antibodies at 4 °C. Sections were washed for 3 × 5 min, then incubated with secondary antibodies for 1 h. Cell nuclei were stained with 4′6-diamidino-2-pheylindole (DAPI).
5
Immunochemical Analysis of Alzheimer's Markers
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Following antibodies were used for this study. AβmOC64 (ab201060, Abcam, MA, USA), Aβ 1-42 (ab10148,), BACE1 (ab63954, Abcam, MA, USA), APP (ab15272, Abcam, MA, USA) CD63 (ab216130, Abcam, MA, USA), CD9 (ab92726, Abcam, MA, USA), Flotillin (ab133497, Abcam, MA, USA), TSG101 (ab125011, Abcam, MA, USA), Alix (ab275377, Abcam, MA, USA), Calnexin (ab133615, Abcam, MA, USA), Argonuate-2 (ab186733, Abcam, MA, USA), GFAP (G3893, Sigma- Aldrich, MO, USA), β-actin (A5316, Sigma- Aldrich, MO, USA), HIF-1α (NB100-449, Novus Biological Company, CO, USA), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, TX, USA) and goat anti-mouse (sc-2005, Santa Cruz Biotechnology, TX, USA), Alexa Fluor 488 conjugated goat anti-mouse (A11001, ThermoFisher Scientific, MA, USA), Alexa Fluor 647 conjugated goat anti-mouse (A32728, ThermoFisher Scientific, MA, USA), Alexa Fluor 594 conjugated goat anti-rabbit (A11012, ThermoFisher Scientific, MA, USA).
6
Immunohistochemical Analysis of Neuroinflammation
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Immunohistochemical staining of GFAP, IbA-1, and Aβ was conducted according to the manufacturer’s protocols and directions. First, antigen-retrieved brain sections were blocked with 3% hydrogen peroxide in methanol for 15 min and then incubated overnight at 4 °C with the primary antibodies (antiglial fibrillary acidic protein as a monoclonal antibody (sc-166458, Santa Cruz Biotechnology, Inc., Dallas, TX, USA, dilution of 1:200), anti-Iba1 antibody (ab108539-Abcam, 1:100), and anti-Aβ antibody (ab201060-Abcam, 1:500)). Next, the sections were washed with PBS three times and incubated with a secondary antibody HRP Envision kit (DAKO) for 20 min. Then, they were washed with PBS three times, incubated with diaminobenzidine for 10 min, counter-stained with Mayer’s hematoxylin, then dehydrated and cleared in xylene. Finally, samples were cover-slipped for microscopic examination.
7
Immunohistochemical detection of Aβ1-42
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The paraffin sections were dewaxed to water, and antigen repair was performed with citrate buffer (PH 6.0, ZLI-9065, ZSBIO, China). Endogenous peroxidase was blocked by 3 % hydrogen peroxide, and goat serum (AR1009, Bosterbio) was used to block at room temperature for 20 min. The primary antibody Aβ1-42 (1:200, ab201060, Abcam, UK) was incubated at 4 °C overnight, and the secondary antibody HRP- labeled goat anti-rabbit IgG (1:100, GB23303, Servicebio, China) was added and incubated at 37 °C for 30 min for immune reaction. Fresh DAB color development solution was developed at room temperature, and the slices were washed with distilled water to terminate color development. Hematoxylin re-stained the nuclei for 3 min and washed with water. The sections were sealed after dehydration and observed under the microscope (BA400Digital, Panthera, China).
8
Immunofluorescence Analysis of Mouse Brain
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After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).
9
Immunofluorescence Analysis of Mouse Brain
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After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).
10
Amyloid-β Peptide Aggregation Assay
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Aβ1−42 was bought from Sigma-Aldrich (St. Louis, USA) and GL Biochem. (Beijing, China) as the lyophilized powder. Hexafluoroisopropanol (HFIP) was purchased from Merck Co. (Darmstadt, Germany). Thioflavin T (ThT), thiazolyl blue tetrazolium bromide (MTT), 8-Anilinonaphthalene-1-sulfonate (ANS), and Hoechst were purchased from Sigma-Aldrich Chem. Co. (St. Louis, USA). The PC12 rat pheochromocytoma cell line was purchased from Pasture Institute (Tehran, Iran). Cell culture plates were acquired from SPL (Beijing, China). Primary antibodies (ab201060 and ab224275) and the secondary antibody (ab6721) were bought from Abcam (Abcam Inc., Cambridge, MA). ECL Plus Kit was purchased from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA). Alexa Fluor 594 (clone Poly4064) was purchased from BioLegend (San Diego, CA, USA). PVDF was purchased from GE Healthcare (Biosciences, Stockholm, Sweden). All other materials were of analytical grade.
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